M.V.
Lomonosov Moscow State University
ANNUAL REPORT
2001
CONTENT
Bioenergetics and photosynthesis
Structure, expression
and evolution of genom
Biology of cell and cell
organelles
Elpidina E.N.,
Vinokurov K.S., Rudenskaya Yu.A., Dunaevsky Ya.E., Zhuzhikov D.P.
Archives of Insect
Biochemistry and Physiology 48 (2001) 217-222
Proteinase inhibitors were studied in the midgut of Nauphoeta
cinerea Oliv. (Blattoptera: Blaberidae) in experimental conditions,
excluding their nutritional origin. One trypsin inhibitor (TI) with Mr
8,000 and two subtilisin inhibitors (SI1 and SI2) with Mr 13,000 and
8,000 were detected after fractionation of total protein preparation on
Sephadex G-50. Ninety-four percent of both types of inhibitors was located in
anterior midgut (AM). TI was 120-fold purified by FPLC-chromatography on Mono
Q. Its isoelectric point was 4.3. TI lost a large part of activity in acidic
and especially in alkaline medium. TI, SI1, and SI2 effectively inhibited
activities of endogenous proteinases from posterior midgut (PM) of the
cockroach. A search for inhibitor of endogenous unusual SH-dependent proteinase
from AM revealed in AM a new inhibitor with Mr 18,000. It was also
inactivated in alkaline medium and was effective against proteinases from PM
along with unusual SH-dependent proteinase from AM. A mechanism of regulation
of activity of midgut proteinases is proposed based on pH-stability of
inhibitors.
Elpidina E.N.,
Vinokurov K.S., Gromenko V.A., Rudenskaya Yu.A., Dunaevsky Ya.E., Zhuzhikov D.P.
Archives of Insect
Biochemistry and Physiology 48 (2001) 206-216
Compartmentalization of proteinases, amylases, and pH in the
midgut of Nauphoeta cinerea Oliv. (Blattoptera:Blaberidae) was
studied in order to understand the organization of protein and starch
digestion. Total proteolytic activity measured with azocasein was maximal at pH
11.5 both in anterior (AM) and posterior (PM) halves of the midgut, but the
bulk of activity (67%) was found in PM. Total AM and PM preparations were
fractionated on a Sephadex G-50 column and further analysed by means of
activity electrophoresis and specific inhibitors and activators. The major
activity in PM was classified as an unusual SH-dependent proteinase with Mr
24,000 and pH optimum with synthetic substrate BApNA at 10.0. The enzyme was
43-fold activated in the presence of 1 mM DTT, insensitive to synthetic
inhibitors of serine (PMSF, TLCK, TPCK) and cysteine (IAA, E-64) proteinases,
strongly inhibited by STI, and displayed four active bands on zymograms. In PM,
activities of trypsin-like, chymotrypsin-like, subtilisin-like, and cysteine
proteinases were observed. Aspartic and metalloproteinases were not detected.
In AM, activity of unusual SH-dependent proteinase also dominated and activity
of chymotrypsin-like proteinase was observed, but their levels were much lower
than in PM. Distribution of amylase activity, exhibiting an optimum at pH 6.0,
was quite the opposite. The major part of it (67%) was located in AM. Treatment
of amylase preparation with proteinases from AM and PM reduced amylase activity
twofold. pH of the midgut contents was 6.0-7.2 in AM, 6.4-7.6 in the first and
8.8-9.3 in the second halves of PM. Thus, pH in AM is in good agreement with
the optimal pH of amylase, located in this compartment, but the activity of
proteinases, including the ability to degrade amylase, in such an environment
is low. Active proteolysis takes place in the second half of PM, where pH of
the gut is close to the optimal pH of proteinases.
Elpidina E.N.,
Rudenskaya Yu.A., Vinokurov K.S., Gromenko V.A., Zhuzhikov D.P.
Journal of
Evolutionary Biochemistry and Physiology 37 (2001) 19-24
The study of proteinase inhibitors in the midgut of the
omnivorous cockroach Nauphoeta cinerea was carried out under conditions
excluding their food origin. One trypsin inhibitor of molecular mass of 8.0 kDa
and three subtilisin inhibitors of molecular masses of 13.0, 8.0, and 4.5 kDa
were found in the protein preparations, using Sephadex G-50 fractionation. 94%
of the activity of the both inhibitor types were located in the anterior midgut
part. Using a high performance liquid chromatography on Mono Q column, the
preparation of trypsin inhibitor was purified 120 times. Its isoelectric point
was to 4.3. The inhibitor lost a part of its activity both under acidic and,
especially, under alkaline conditions and was completely inactivated at pH 10.
The studied inhibitors inhibited effectively activities of trypsin-like and
subtilisin-like proteinases from the cockroach posterior midgut part. The
possible physiological role of the proteinase inhibitors and, particularly,
their partici pation in regulation of digestion in the midgut of N. cinerea are
discussed.
Tsybina T.A.,
Dunaevsky Y.E., Musolyamov A.K., Egorov T.A., Belozersky M.A.
Biochemistry (Moscow)
66 (2001) 941-947
Preparations of low molecular weight protein inhibitors of
serine proteinases have been obtained from buckwheat (Fagopyrum esculentum)
seeds by chromatography of seed extract on trypsin-Sepharose 4B, Mono-Q, and
Mono-S ion exchangers (FPLC regime). Their molecular masses, determined by mass
spectrometry, were 5203 (BWI-1c), 5347 (BWI-2c), 7760 (BWI-3c), and 6031
daltons (BWI-4c). All of the inhibitors possess high pH- and thermal stability
in the pH range 2-12. In addition to trypsin, BWI-3c and BWI-4c inhibited
chymotrypsin and subtilisin-like bacterial proteases. The N-terminal sequences
of all of the inhibitors were determined: BWI-1c (23 residues), BWI-2c (33
residues), BWI-3c (18 residues), and BWI-4c (20 residues). In their
physicochemical properties and N-terminal amino acid sequences, the buckwheat
seed trypsin inhibitors BWI-3c and BWI-4c appear to belong to potato proteinase
inhibitor I family.
Sukhomlinova
M.Yu., Kireyev I.I., Fais D., Giudice G., Polyakov V.Yu.
Membrane
& Cell Biology 14 (2001) 605-615
The dynamics of structural changes of the
chondriome in the early development of the sea urchin Paracentrotus lividus
was studied. Mature eggs and embryos at various stages of cleavage were used
for quantitative and ultrastructural analysis based on computerized 3D
reconstruction from serial ultrathin sections. The following structural
transformations of the chondriome were shown to occur in the course of
embryogenesis: (i) 15 min after fertilization, mitochondrial clusters
disintegrate, and mitochondrial division is induced. At the stage of two
blastomeres the population of mitochondria increases twofold; (ii) the
mitochondria divide by means of the contraction of both outer and inner
membranes. The forming furrow divides the "parental" mitochondrion
into two equal "daughter" parts; (iii) at the four-cell stage the
division ceases, and mitochondria start to grow, so that the mitochondrial
length increases; (iv) cell differentiation further stimulates elongation of
rod-shaped mitochondria, and the ratio of rod-shaped to spherical mitochondria
changes; (v) in an unfertilised egg, the mitochondria are in a condensed form;
after fertilisation all the mitochondria acquire a conventional form. Modern
concepts of chondriome proliferation in eukaryotic cells are discussed.
Sheval’
E.V., Gulak P.V., Kireev I.I., Chudinova E.M., Fais D., Dzhanguzza F., Polyakov
V.Yu.
Russian
Journal of Developmental Biology 32 (2001) 313-319
The structure of a “noncanonical” nucleolus of
vitellogenic oocytes in the sea urchin Paracentrotus lividus was studied
using the inhibitor of transcription actinomycin D. In the control cells, the
nucleolus consists of two separated structural subdomains: the dense
fibrillar-granular peripheral area and the fibrillar central area. The
nucleolus did not contain subdomains corresponding to the fibrillar center and
dense fibrillar component of “typical” nucleoli. After treatment with
actinomycin D, numerous argyrophilic granules appeared in the karyoplasm, the
intranucleolar DNA became compact, and the nucleolar material was segregated
into two or three separated zones, the residual peripheral area being the
densest and largest. Lesser zones had a decreased electron density and
contained argyrophilic proteins and, apparently, the nucleolar organizer
material. These results suggest that, for normal rRNA expression and
processing, the presence of structural subdomains in the nucleolus, such as
fibrillar complexes and a dense fibrillar component is not essential.
Muntz K.,
Belozersky M.A., Dunaevsky Ya.E., Schlereth A., Tiedemann J.
Journal
of Experimental Botany 52 (2001) 1741-1752
Though endopeptidases and carboxypeptidases are
present in protein bodies of dry quiescent seeds the function of these
proteases during germination is still a matter of debate. In some plants it was
demonstrated that endopeptidases of dry protein bodies degrade storage proteins
of these organelles. Other studies describe cases where this did not happen.
The role that stored proteinases play in the initiation of storage protein
breakdown in germinating seeds thus remains unclear. Numerous reviews state
that the initiation of reserve protein mobilization is attributed to de novo formed
endopeptidases which together with stored carboxypeptidases degrade the bulk of
proteins in storage organs and tissues after seeds have germinated. The
evidence that the small amounts of endopeptidases in protein bodies of
embryonic axes and cotyledons of dry seeds from dicotyledonous plants play an
important role in the initiation of storage protein mobilization during early
germination is summarized here.
Kobliakova
Yu.V., Kireev I.I., Stefanova V.N., Zatsepina O.V., Poliakov V.Yu.
Tsitologiia
43 (2001) 462-470
A new method of differential decondensation of
mitotic chromosomes has been proposed by means of repeated treatment of live
cells with 15% Hanks' balanced salt solution. The procedure of cell treatment
includes three stages: the first hypotonic shock, cultivation in isotonic
medium, and the second hypotonic shock. As a result, after a standard
methanol-acetic acid fixation and Giemsa staining some discrete Giemsa-positive
globules are revealed in mitotic chromosomes. Such globules are symmetrically
arranged in axial regions of sister chromatids. The comparative analysis of
marker chromosomes has revealed a topological conformity of these globules to
G-bands of chromosomes. It has been shown that it is the first hypotonic shock
that triggers induction of structural modification of chromatin in interphase
nuclei and in mitotic chromosomes. Of interest is the fact that the effect of
the first shock is prolonged in time and is realized during at least one cell
cycle, with the normal structure of mitotic chromosomes being restored after
S-phase of the successive cell cycle.
Pavlova
G.A.
The
Journal of Experimental Biology 204 (2001) 1625-1633
The terrestrial snail Helix lucorum
crawls using waves of muscular contraction (pedal waves) that spread along the
sole of its foot. Crawling speed depends on wave generation frequency (step
frequency) and the distance the snail moves forwards during each wave (step
length). In a previous study, video recordings of a crawling snail showed that
its sole length varied over a wide range and was directly correlated
withmollusc speed. Speed depended on step length, which was directly related to
solelength, rather than on step frequency, which remained rather constant. In
the present study, the effects of dopamine, ergometrine (a blocker of dopamine receptors
in molluscs) and serotonin injection on the linear relationship between sole
length and locomotor speed in Helix lucorum were studied. In crawling
snails, dopamine caused sole contraction, and locomotion slowed down or ceased.
Ergometrine stimulated locomotion, which resembled rapid crawling with an
extended sole, as observed under normal conditions. Serotonin stimulated
locomotion and accelerated crawling significantly without causing changes in
sole length. The acceleration of locomotion induced by serotonin injection was
due to pedal wave (step) elongation. It is proposed that, during each locomotor
episode, dopamine controls snail speed by regulating sole length, which
determines the amplitude of contraction of the muscle cells involved in pedal
waves and, as a result, step length; serotonin determines the basic step length
and shifts the linear relationship between sole length and mollusc speed
upwards along the axis of mollusc speed. The efficiency of the serotonergic
system depends on the physiological state of the mollusc (e.g. that
characteristic of summer or winter).
Barsukova
A.S., Artemenko E.G., Kalaidzidis A.L., Zatsepina O.V.
Tsitologiia
43 (2001) 46-51
It is known that in mice the centromeric
heterochromatin remains compact during the whole cell cycle and at interphase
is referred to as "chromocentres". In the current study, by the use
of antibodies against prekinetochores and DNA polymerase (a PCNA antigen), we
showed that in murine L929 cells chromocentres remain spatially associated with
prekinetochores during the entire interphase, including the late S-period, when
DNA chromocentres replicate. Augmentation of prekinetochore fluorescence
increases concomitantly with the heterochromation replication, but the
prekinetochore duplication occurs only in G2 period. A conclusion has been made
that murine interphase cells can be used for biochemical fractionation of
chromocentres associated with prekinetochore proteins.
Domnina
L.V., Ivanova O.Yu., Vasil'ev Yu.M.
Tsitologiia
43 (2001) 133-141
The aim of this work was to study role of the contractility
in the process of fibroblast spreading. We investigated the morphology and
cytoskeleton of cells seeded in the medium containing 2,3 butanedione monoxime
(BDM), an inhibitor of myosin II and myosin-ATPase. Time-lapse video
observation and immunofluorescence microscopy were used. BDM caused delay in
spreading and blocked cell polarization, that led eventually to the
conservation of disk-like cell morphology. The actin-myosin cytoskeleton was
also BDM-changed. The number and thickness of stress-fibers decreased. Myosin
II orientation was dramatically disturbed to obtain a difuse pattern in the
cytoplasm. Paxillin-containing focal adhesions decreased in length and their
distribution was changed. The movement of concanavalin A receptors and concanavalin
A-coated beads on the lamellar cell surface was also BDM inhibited. It
indicates an obvious depression of the lamellar cytoplasm activity and points
to the damage of the actin-myosin cytoskeleton. Thus, the change in
contractility of the latter alters significantly the morphogenesis of
fibroblast spreading.
Pletjushkina
O.J., Rajfur Z., Pomorski P., Oliver T.N., Vasiliev J.M., Jacobson K.A.
Cell
Motil Cytoskeleton 48 (2001) 235-244
Actomyosin-based cortical contractility is a
common feature of eukaryotic cells but the capability to produce rhythmic
contractions is found in only a few types such as cardiomyocytes. Mechanisms
responsible for the acquisition of this capability remain largely unknown.
Rhythmic contractility can be induced in non-muscle cells by microtubule
depolymerization. Spreading epithelial cells and fibroblasts in which
microtubules were depolymerized with nocodazole or colcemid underwent rhythmic
oscillations of the body that lasted for several hours before the cells
acquired a stable, flattened shape. By contrast, control cells spread and
flattened into discoid shapes in a smooth and regular manner. Quantitative
analysis of the oscillations showed that they have a period of about 50
seconds. The kinase inhibitors, HA 1077 and H7, and the more specific
rho-kinase inhibitor, Y 27632, caused the oscillations to immediately cease and
the cells to become flat. Transient increases in cytoplasmic calcium preceded
the contractile phase of the oscillations. Wrinkle formation by cells plated on
elastic substrata indicated that the contractility of colcemid-treated cells
increased in comparison to controls but was drastically decreased after HA 1077
addition. These data suggest that an intact microtubular system normally
prevents pulsations by moderating excessive rho-mediated actin myosin
contractility. Possible mechanistic interactions between rho-mediated and
calcium activated contractile pathways that could produce morphological
oscillations are discussed.
Kaspieva O.V.,
Nikolaeva O.P., Orlov V.N., Ponomarev M.A., Drachev V.A., Levitsky D.I.
FEBS
Letters 489 (2001) 144-148
Differential scanning calorimetry (DSC) was used
to analyze the thermal unfolding of myosin subfragment 1 (S1) with the SH1
(Cys-707) and SH2 (Cys-697) groups cross-linked by N,N'-p-phenylenedimaleimide
(pPDM-S1). It has been shown that F-actin affects the thermal unfolding of
pPDM-S1 only at very low ionic strength, when some part of pPDM-S1 binds weakly
to F-actin, but not at higher ionic strength (200 mM KCl). The weak binding of
pPDM-S1 to F-actin shifted the thermal transition of pPDM-S1 by about 5 degrees
C to a higher temperature. This actin-induced increase in thermal stability of
pPDM-S1 was similar to that observed with 'strong' binding of unmodified S1 to
F-actin. Our results show that actin-induced structural changes revealed by DSC
in the myosin head occur not only upon strong binding but also on weak binding
of the head to F-actin, thus suggesting that these changes may occur before the
power-stroke and play an important role in the motor function of the head.
Appearance
of "b-like" circular dichroism spectra on protein
aggregation that is not accompanied by transition to b-structure.
Arutyunyan
A.M., Rafikova E.R., Drachev V.A., Dobrov E.N.
Biochemistry
(Moscow) 66 (2001) 1378-1380
CD spectra in the 200 to 250 nm spectral region
for small ordered aggregates (trimers-pentamers) of tobacco mosaic virus (TMV)
coat protein (CP) and for long virus-like helical aggregates of TMV CP were compared.
It was found that small (4S) TMV CP aggregates have a CD spectrum typical of a
protein with high a-helix content, which agrees well with results
of X-ray diffraction studies. But in the long helical aggregates (and in the
TMV virions) TMV CP gives "b-like" CD spectra similar to those of many other aggregated
proteins. From X-ray diffraction data, it is well known that TMV CP subunits do
not change their secondary or tertiary structure on assembly into virions or
the helical repolymerized protein. Thus, the change in the shape of 200 to 250
nm CD spectra cannot be employed as the sole criterion of the conversion of a
protein to b-structure in the course of aggregation.
Mukhar'iamova
K.Sh, Zatsepina O.V.
Tsitologiia
43 (2001) 792-796
We applied a sensitive and specific method for
detection of run-on rDNA transcription in cultured mammalian cells. This
technique is based on the capability of RNA polymerase I to maintain
transcriptional activity following cell fixation with methanol, and on the use
of BrUTP as a precursor of rRNA synthesis. The results obtained have shown that
in cultured pig cells (PK cells) the ribosomal genes are transcribed during
interphase to become repressed at the end of mitotic prophase. The rDNAs are
not transcribed at the prometaphase, metaphase and anaphase stages. The
ribosomal genes become derepressed at early telophase. At early telophase, the
number of BrUTP-incorporated sites is equal to that of the nucleolus organizing
regions (NORs), but it is augmented during telophase progression. A similar
dynamics of ribosomal gene reactivation is also revealed following spatial
separation of NO-chromosomes between individual micronuclei caused by hypotonic
chock. This indicates that the spatial integration of chromosomal NORs is not a
prerequisite for ribosomal gene reactivation at mitosis.
Sheval'
E.V., Prusov A.N., Kireev I.I., Poliakov V.Yu.
Tsitologiia
43 (2001) 122-132
The role of the nuclear matrix components in the
organization of structural and functional domains of interphase nuclei was
studied using irradiation with blue light in the presence of a
photosensibilized agent (Ethidium bromide). Nuclear domain resistance to
extractive solution (2M NaCl) treatment served as a criterion of
irradiation-induced stabilization of different nuclear domains. The following results
have been obtained: 1) the structural organization of the complexes of
chromatin and clusters of replication does not depend on the state of the
nuclear matrix in isolated nuclei; 2) chemical stabilization of the nuclear
matrix by Cu2+-ions is not sufficient for the organization of
chromatin domains; 3) irradiation in the presence of Ethidium bromide
stabilizes domains of the nuclei, but does not lead to stabilization of the
nuclear matrix internal network. Hence, the irradiation prevented extraction
from the nuclear domains of nonhistone proteins, which were not standard matrix
proteins. Based on the results obtained, a hypothesis was proposed about a
coexistence of two groups of nonhistone proteins in the cell nucleus. The first
group includes proteins of the nuclear matrix involved in immobilization of
scafford attachment regions and active genes. The second group includes some
hypothetical structural proteins participating only in compaction of DNA of
condensed chromatin.
Barsukova
A.S., Fedorova N.E., Medzhidova A.A., Zatsepina O.V., Kushch A.A.
Russian
Journal of Developmental Biology 32 (2001) 29-34
The effect of cytomegalovirus on the cell cycle
was studied autoradiographically in an asynchronous culture of human diploid
fibroblasts. The analysis of labeled mitosis showed that some cells infected in
the S phase ceased to progress through the cell cycle at one of its phases (S,
G2, or M); at the same time, at least part of infected cells remained capable
of entering mitosis. Beginning from day 2 after infection by cytomegalovirus,
the accumulation of pathological mitotic cells blocked at metaphase was observed
in the culture. Approximately 50% of these cells contained 3H-thymidine label
above chromosomes. This fact suggested the possibility of pathological mitosis
in cells that were infected both at the S and other phases of the cell cycle.
The detailed morphological analysis of chromosomes at different stages of
infection demonstrated that the degree of their morphological changes increases
from slight (stronger condensation) to severe pathology (fragmentation). In the
aggregate, the results of the study suggested that abnormal chromosome
morphology resulted from irreversible cell division arrest under the effect of
cytomegalovirus.
Vorobjev I.,
Malikov V., Rodionov V.
Proc.
Natl. Acad. Sci. USA 98 (2001) 10160-10165
Polarized radial arrays of cytoplasmic
microtubules (MTs) with minus ends clustered at the cell center define the
organization of the cytoplasm through interaction with microtubule motors bound
to membrane organelles or chromosomes. It is generally assumed that the radial
organization results from nucleation of MTs at the centrosome. However, radial
MT array can also be attained through self-organization that requires the activity
of a minus-end-directed MT motor, cytoplasmic dynein. In this study we examine
the role of cytoplasmic dynein in the self-organization of a radial MT array in
cytoplasmic fragments of fish melanophores lacking the centrosome. After
activation of dynein motors bound to membrane-bound organelles, pigment
granules, the fragments rapidly form polarized radial arrays of MTs and
position pigment aggregates at their centers. We show that rearrangement of MTs
in the cytoplasm is achieved through dynein-dependent MT nucleation. The radial
pattern is generated by continuous disassembly and reassembly of MTs and
concurrent minus-end-directed transport of pigment granules bearing the
nucleation sites.
Chernobel'skaia
O.A., Grigor'ev I.S., Alieva I.B., Vorob'ev I.A.
Russian
Journal of Developmental Biology32 (2001) 58-66
It is generally assumed that microtubules in
tissue culture cells extend from the centrosome to cell periphery, and the
length of individual microtubules averages several dozens of microns. However,
direct electron-microscopic measurements have cast some doubt on this
assumption. In this study, the average length of microtubules in cultured Vero
cells was estimated using a combined approach. The length of free cytoplasmic
and centrosomal microtubules was determined by means of electron microscopy in
serial sections; concurrently, the length of free microtubules in the lamella
was measured in preparations stained with tubulin antibodies (an indirect
immunofluorescent method), by tracing saltatory particle movements along the
microtubules in living cells. According to the data of immunofluorescent
microscopy, microtubule length in the lamella averaged 4.57 ± 3.69 microns. However, since two or more microtubules can
overlap, their length may be slightly overestimated by this method. On the
other hand, saltatory movements are easy to monitor and measure fairly
accurately, but their range may be shorter than the actual microtubule length
because of a limited processiveness of motors (kinesin and dynein). On average,
the trajectories of saltatory movements in living cells were 3.85 ± 0.72 microns long. At the electron-microscopic level, microtubule
length was analyzed using pseudo-three-dimensional reconstructions of the
microtubule systems around the centrosome and in the lamella. The length of
free microtubules in the lamella reached 18 microns, averaging 3.33 ± 2.43 microns; the average length of centrosomal microtubules was
1.49 ± 0.82 microns. Good correspondence between the
data on microtubule length and arrangement obtained by different methods allows
the conclusion that most of free microtubules in Vero cells actually have a
length of 2-5 microns; i.e., they are much shorter than the cell radius (about
25 microns). Microtubules extending from the centrosome are shorter still and
do not reach the cell periphery. Thus, most microtubules in the lamella of Vero
cells are free and their ordered arrangement is not associated with their
attachment to the centrosome.
Omelchenko
T., Fetisova E., Ivanova O., Bonder E.M., Feder H., Vasiliev J.M., Gelfand I.M.
Proc.
Natl. Acad. Sci. USA 98 (2001) 8632-8637
Contact interactions between different cell
types play a number of important roles in development, for example in cell
sorting, tissue organization, and ordered migration of cells. The nature of
such heterocellular interactions, in contrast to interactions between cells of
the same type, remains largely unknown. In this report, we present experimental
data examining the dynamics of heterocellular interactions between
epitheliocytes and fibroblasts, which express different cadherin cell adhesion
molecules and possess different actin cytoskeletal organizations. Our analysis
revealed two striking features of heterocellular contact. First, the active
free edge of an epitheliocyte reorganizes its actin cytoskeleton after making
contact with a fibroblast. Upon contact with the leading edge of a fibroblast,
epitheliocytes disassemble their marginal bundle of actin filaments and
reassemble actin filaments into a geometric organization more typical of a
fibroblast lamella. Second, epitheliocytes and fibroblasts form cell-cell
adhesion structures that have an irregular organization and are associated with
components of cell adhesion complexes. The structural organization of these
adhesions is more closely related to the type of contacts formed between
fibroblasts rather than to those between epitheliocytes. Heterotypic
epithelio-fibroblastic contacts, like homotypic contacts between fibroblasts,
are transient and do not lead to formation of stable contact interactions. We
suggest that heterocellular contact interactions in culture may be regarded as
models of how tissue systems consisting of epithelia and mesenchyme interact
and become organized in vivo.
Dugina
V., Fontao L., Chaponnier C., Vasiliev J., Gabbiani G.
Journal
of Cell Science 114 (2001) 3285-3296
Transforming growth factor b (TGFb), the most established promoter of
myofibroblast differentiation, induces ED-A cellular fibronectin and a-smooth muscle actin expression in fibroblastic cells in vivo and
in vitro. ED-A fibronectin exerts a permissive action for a-smooth muscle actin expression. A morphological continuity
(called fibronexus), a specialized form of focal adhesion, has been described
between actin stress fibers that contain a-smooth muscle actin, and extracellular
fibronectin, which contains the ED-A portion, in both cultured fibroblasts and
granulation tissue myofibroblasts. We have studied the development of these
focal adhesions in TGFb-treated fibroblasts using confocal laser
scanning microscopy, three-dimensional image reconstruction and western blots
using antibodies against focal adhesion proteins. The increase in ED-A
fibronectin expression induced by TGFb was accompanied by bundling of ED-A fibronectin
fibers and their association with the terminal portion of a-smooth muscle actin-positive stress fibers. In parallel, the
focal adhesion size was importantly increased, and tensin and FAK were
neoexpressed in focal adhesions; moreover, vinculin and paxillin were recruited
from the cytoplasmic pool into focal adhesions. We have evaluated
morphometrically the length and area of focal adhesions. In addition, we have
evaluated biochemically their content of associated proteins and of a-smooth muscle actin after TGFb stimulation and on this basis suggest a new
focal adhesion classification, that is, immature, mature and supermature. When
TGFb-induced a-smooth muscle actin expression was blocked by
soluble recombinant ED-A fibronectin, we observed that the fragment was
localised into the fibronectin network at the level of focal adhesions and that
focal adhesion supermaturation was inhibited. The same effect was also exerted
by the ED-A fibronectin antibody IST-9. In addition, the antagonists of
actin-myosin contractility BDM and ML-7 provoked the dispersion of focal
adhesions and the decrease of a-smooth muscle actin content in stress fibers of
pulmonary fibroblasts, which constitutively show large focal adhesions and
numerous stress fibers that contain a-smooth muscle actin. These inhibitors also
decreased the incorporation of recombinant ED-A into fibronectin network. Our
data indicate that a three-dimensional transcellular structure containing both
ED-A fibronectin and a-smooth muscle actin plays an important role in
the establishment and modulation of the myofibroblastic phenotype. The
organisation of this structure is regulated by intracellularly and
extracellularly originated forces.
Nadezhdina E.S., Zinovkina
L.A., Fais D., Chentsov Y.S.
Russian Journal of
Developmental Biology32 (2001) 41-50
We
studied the possibility of using the spermatozoa of the loach Misgurnus
fossilis L. for identification of centrosome proteins. It has been shown that
the centrosome of the loach spermatozoa consists of a pair of centrioles of the
standard structure and contains the marker protein g-tubulin, cytoplasmic microtubules
branch out from it, and it does not contain any additional structures
characteristic of the centrosomes of spermatozoa of many other fishes. A preparation
enriched with intact centrosomes has been obtained from the loach spermatozoa.
These centrosomes contained g-tubulin although they lost their
ability to induce polymerization of microtubules. The preparation of loach
centrosomes was successfully used to obtain a set of monoclonal antibodies
against the mammalian centrosome. A new protein kinase LOSTEK was identified
with the help of one of these monoclonal antibodies, SN2-3D2, which was
localized in the centrosome and on then microtubules in both loach spermatozoa
and cultured mammalian cells. Hence, the loach spermatozoa are a promising
object for identification of new proteins of the mammalian centrosome.
Shanina N.A., Ivanov P.A.,
Chudinova E.M., Severin F.F., Nadezhdina E.S.
Molecular Biology 35 (2001)
638-646
Association
of the translation apparatus with the cytoskeleton is essential for its transportation
within the cell and probably also for translation regulation. Very little is
known about the involvement of particular proteins of this association. A
polypeptide homologous with the heavy chain of translation initiation factor
eIF3 p170 was found earlier in a microtubule preparation from adrenal cells.
Antibody A167 directed against the recombinant fragment of p170 has been
generated to study eIF3 interaction with microtubules in mammalian cells. This
antibody was shown to recognize a single 170 kDa polypeptide in eIF3
preparations as well as in homogenates of various cell types. A167 allowed
detection of the 170 kDa polypeptide in microtubule preparation from bovine
brain and confirmation of its presence in microtubule preparations from adrenal
cells. As shown by immunofluorescence microscopy using A167, the 170 kDa
polypeptide is mainly located in the endoplasm within numerous small and some
large granules. Cell treatment with cycloheximide resulted in growth and
clustering of the large granules, and partial antigen redistribution along
cellular microtubules. These new experimental data indicate that mammalian
translation factor eIF3 may bind with microtubules.
Belgareh N., Rabut G., Bai
S.W., van Overbeek M., Beaudouin J., Daigle N., Zatsepina O.V., Pasteau F.,
Labas V., Fromont-Racine M., Ellenberg J., Doye V.
The Journal of Cell Biology
154 (2001) 1147-1160
The
nuclear pore complexes (NPCs) are evolutionarily conserved assemblies that
allowtraffic between the cytoplasm and the nucleus. In this study, we have
identified and characterized a novel human nuclear pore protein, hNup133,
through its homology with the Saccharomyces cerevisiae nucleoporin scNup133.
Two-hybrid screens and immunoprecipitation experiments revealed a direct and
evolutionarily conserved interaction between Nup133 and Nup84/Nup107 and
indicated that hNup133 and hNup107 are part of a NPC subcomplex that contains two
other nucleoporins (the previously characterized hNup96 and a novel nucleoporin
designated as hNup120) homologous to constituents of the scNup84 subcomplex. We
further demonstrate that hNup133 and hNup107 are localized on both sides of the
NPC to which they are stably associated at interphase, remain associated as
part of a NPC subcomplex during mitosis, and are targeted at early stages to
the reforming nuclear envelope. Throughout mitosis, a fraction of hNup133 and
hNup107 localizes to the kinetochores, thus revealing an unexpected connection
between structural NPCs constituents and kinetochores. Photobleaching
experiments further showed that the mitotic cytoplasm contains
kinetochore-binding competent hNup133 molecules and that in contrast to its
stable association with the NPCs the interaction of this nucleoporin with
kinetochores is dynamic.
Zelenin P.V., Grillner S.,
Orlovsky G.N., Deliagina T.G.
The Journal of Neuroscience
21 (2001) 7793-7803
The
effects of signals transmitted from the brain to the spinal locomotor networks
by a population of command neurons are determined by specific functional
projections of each individual neuron. To reveal these projections, we used a
simple vertebrate model, the lamprey, in which responses of the spinal networks
to spikes in single reticulospinal axons were detected by using the
spike-triggered averaging of the motoneuronal activity. We found that
individual neurons exert a uniform effect on the segmental motor output along
the whole extent of their axons. Twenty different patterns of effect, that is,
combinations of influences on the segmental motoneuron pools, were found. The
widespread projections and heterogeneity of the population of command neurons
present a basis for formation of different gross motor synergies.
Fagerstedt P., Orlovsky
G.N., Deliagina T.G., Grillner S., Ullen F.
Journal of Neurophysiology
86 (2001) 2257-2265
We
studied the neural correlates of turning movements during fictive locomotion in
a lamprey in vitro brain-spinal cord preparation. Electrical stimulation
of the skin on one side of the head was used to evoke fictive turns.
Intracellular recordings were performed from reticulospinal cells in the middle
(MRRN) and posterior (PRRN) rhombencephalic reticular nuclei, and from Mauthner
cells, to characterize the pattern of activity in these cell groups, and their
possible functional role for the generation of turns. All recorded
reticulospinal neurons modified their activity during turns. Many cells in both
the rostral and the caudal MRRN, and Mauthner cells, were strongly excited
during turning. The level of activity of cells in rostral PRRN was lower, while
the lowest degree of activation was found in cells in caudal PRRN, suggesting
that MRRN may play a more important role for the generation of turning
behavior. The sign of the response (i.e., excitation or inhibition) to skin
stimulation of a neuron during turns toward (ipsilateral), or away from
(contralateral) the side of the cell body was always the same. The cells could
thus be divided into four types: 1) cells that were excited during ipsilateral
turns and inhibited during contralateral turns; these cells provide an
asymmetric excitatory bias to spinal networks and presumably play an important
role for the generation of turns; these cells were common (n = 35; 52%) in both
MRRN and PRRN; 2) cells that were excited during turns in either direction;
these cells were common (n = 19; 28%), in particular in MRRN; they could be
involved in a general activation of the locomotor system after skin stimulation;
some of the cells were also more activated during turns in one direction and
could contribute to an asymmetric turn command; 3) one cell that was inhibited
during ipsilateral turns and excited during contralateral turns; and 4) cells
(n = 12; 18%) that were inhibited during turns in either direction. In summary,
our results show that, in the lamprey, the large majority of reticulospinal
cells have responses during lateral turns that are indicative of a causal role
for these cells in turn generation. This also suggests a considerable overlap
between the command system for lateral turns evoked by skin stimulation, which
was studied here, and other reticulospinal command systems, e.g., for lateral
turns evoked by other types of stimuli, initiation of locomotion, and turns in
the vertical planes.
Kozlov A.K., Aurell E.,
Orlovsky G.N., Deliagina T.G., Zelenin P.V., Hellgren-Kotaleski J., Grillner S.
Biological Cybernetics 84
(2001) 323-330
A
phenomenological model of the mechanism of stabilization of the body
orientation during locomotion (dorsal side up) in the lamprey is presented. The
mathematical modeling is based on experimental results obtained during
investigations of postural control in lampreys using a combined in vivo
and robotics approach. The dynamics of the model agree qualitatively with the
experimental data. It is shown by computer simulations that postural correction
commands from reticulospinal neurons provide information sufficient to
stabilize body orientation in the lamprey. The model is based on differences
between the effects exerted by the vestibular apparatus on the left and the
right side.
Gorbushin
A.M., Levakin I.A., Panchina N.A., Panchin Y.V.
The Journal of Experimental
Biology 204 (2001) 283-289
Within
2 weeks of decapitation, Hydrobia ulvae was able to regenerate new head
structures including buccal ganglia. It was also capable of regenerating
propodial ganglia after anterior foot amputation. The functional regeneration
of the buccal ganglia was demonstrated by behavioural observations and by
electrophysiological experiments. The presence of the oesophagus was shown to
be important for regeneration of the buccal complex. H. ulvae provides a
new model for regeneration studies, so details of the topographic anatomy and
biology of this species are described. To standardize experimental animals in
future studies, the effects of age, sex and trematode infestation on the
regeneration capacity of H. ulvae have been evaluated. The high capacity
for regeneration together with the possibility of using electrophysiological
techniques makes H. ulvae a favourable model in which to study
neurogenesis in adult animals.
Bogacheva A.M., Rudenskaya
G.N., Dunaevsky Y.E., Chestuhina G.G., Golovkin B.N.
Biochimie 83 (2001) 481-486
A
new subtilisin-like proteinase hydrolyzing chromogenic peptide substrate
Glp-Ala-Ala-Leu-p-nitroanilide optimally at pH 8.1 was found in common plantain
leaves. The protease named plantagolisin was isolated by ammonium sulfate
precipitation of the leaves' extract followed by affinity chromatography on
bacitracin-Sepharose and ion-exchange chromatography on Mono Q in FPLC regime.
Its molecular mass is 19000 Da and pI 5.0. pH-stability range is 7-10 in the
presence of 2 mM Ca2+, temperature optimum is 40 degrees C. The
substrate specificity of subtilase towards synthetic peptides and insulin
B-chain is comparable with that of two other subtilisin-like serine
proteinases: proteinase from leaves of the sunflower and taraxalisin. Besides,
the proteinase is able to hydrolyze substrates with Pro in P1
position. The enzyme hydrolyzes collagen. a and b chains are hydrolyzed
simultaneously in parallel; there are only low-molecular-mass hydrolysis
products in the sample after 2 h of incubation. Pure serine proteinase was
inactivated by specific serine proteinases inhibitors: diisopropylfluorophosphate,
phenylmethylsulfonyl fluoride and Hg2+. The plantagolisin N-terminal
sequence ESNSEQETQTESGPGTAFL-, traced for 19 residues, revealed 37% homology
with that of subtilisin from yeast Schizosaccharomyces pombe.
Archambault P.S., Deliagina
T.G., Orlovsky G.N.
Neuroreport 12 (2001)
1803-1807
The lamprey (a lower vertebrate,
cyclostome), in addition to ordinary swimming, is also capable of crawling.
Here we describe crawling forward in a narrow U-shaped tunnel. A rapid movement
along the tunnel was evoked by stimulating the tail. The muscle activity
responsible for propulsion was confined to the area around the body bend.
Muscles on the inner (concave) side were activated when approaching the turn,
and inactivated on the top of the arc. Muscles on the outer (convex) side were
co-active with their antagonists, but also active in the area of straightening
of the body bend. This pattern of muscle activity propagated along the body.
The role of central and reflex mechanisms in the generation of locomotor
movements is discussed.
Ivanova T.V., Ivanov V.N.,
Nadezhdina E.S.
Membrane & Cell Biology
14 (2001) 727-741
The effects of nocodazole (an agent
causing degradation of microtubules in vitro) on the content of
transcription factors NF-kB and AP-1/c-fos in the nuclei of cultured cells
CHO-K1 and HeLa, as well as on the activity of these factors in the nuclei of
T-cell hybridoma 2B4 were studied. Immunoblotting, immunofluorescence, and gel
retardation techniques revealed that in all cell lines studied nocodazole
induced rapid and considerable activation (an increased content in the nuclei)
of transcription factor AP-1/c-fos. In 2B4 cells, nocodazole had no effect on
the activity of NF-kB, but in CHO-K1 and HeLa cells it caused a transient
decrease, sometimes followed by an increase in the content of subunit p50. Besides,
nocodazole inhibited proteolytic degradation of NF-kB in CHO-K1 and HeLa cells and
caused a short-term decrease in the content of subunit p65 in CHO-K1 cells. The
results suggest that nocodazole may interfere with the genome expression, and
these alterations should be taken into consideration, whatever experimental
studies this substance is used in.
Cyclosporin
A Blocks PMA and lonomycin Activated LymphotoxinExpression in a Human T-Cell
Line
Boitchenko
V., Kuprash D., Nordheim A., Ruhlmann A., Nedospasov S.A.
Russian
Journal of Immunology 6 (2001) 9-
LTa and LTb represent subunits of lymphotoxin complexes,
forming either a soluble homotrimeric complex (LTa3), which binds to and signals through CD120a/b
(TNFRp55 and p75), or a membrane-associated heterotrimeric complex (LTa1b2), which binds to and signals through the LTb receptor (LTbR). While it was demonstrated that the LTa1b2 complex is essential for organogenesis and the
functional organization of secondary lymphotd organs, regulation of the biosynthesis
of the LT components is not fully understood. We studied regulation of LTa and LTb gene expression in human T-cell line MOLT4,
with special emphasis on the modulatory effects of the immunosuppressant
cyclosporin A (CsA). CsA downregulates PMA/ionomycin-induced LTa at both transcription and protein expression levels, whereas it
downregulates LTb at the transcript but not at the protein
expression levels. Our data suggest that intracellular calcium homeostasis
determines the balance of LTa and LTb in human T cells.
Potapova
T.V., Boytzova L.Yu., Alexeevski A.V., Smolianinov V.V.
Biological
Membranes (Moscow) 18 (2001) 389-394
An
experimental model of a hyphal mini-tree was developed for Neurospora crassa
filamentous fungi to estimate quantitatively the dynamics of changes in the
parameters of polarized tip growth. The electric and diffusional coupling of
the hyphal apex with distant mycelium zones has been disrupted by the
microelectrode damage of individual hyphal compartment at the 500-700 mm from the apex. The 30 min growth of the isolated mini-trees
resulted to form the trunk length near 900 mm and diminish twice the rate leading tip
elongation as well as number of side branch formation. The hyphal growth unit
(the ratio of the total length of all the branches to the number of the tips)
was shown to be independent of the coupling with the mycelium. The elongation
rate of the leading tip of an isolated 500 mm hyphal "mini-tree" changes
immedeately after isolation, that one of the 700 mm "mini-tree" does not differ from that of intact hyphae
for the first 20 min after uncoupling. These data allow to estimate
quantitativly the matter input from the mycelium. When the distance from the
branch tip of the mini-tree to the site of electrical uncoupling was less than
450 mm, its growth stopped.
Shakhov
A.N., Nedospasov S.A.
Cytokine
Growth Factor Review 12 (2001) 107-119
Expression profiling provides a powerful
approach to define the underlying molecular mechanisms in disease. Several
techniques referred collectively to as gene profiling may be also helpful in
the analysis of the phenotype of mice with targeted mutations, especially if
applied to distinct histological compartments, to specific cell types or to
evaluate the effect of specific challenges, such as infection. Here we review
several of the existing techniques applicable to genetic knockout studies, and
share our experience from the study of mice with tumor necrosis factor (TNF)
and lymphotoxin (LT) deficiencies, with specific emphasis on the distinction
between TNF- and LT-mediated signalling pathways in vivo. Gene
expression profiling analysis of TNF/LT-deficient mice supports the notion that
TNF and LT, originally discovered as distinct biological activities, manifest
both distinct and redundant functions in vivo.
Boitchenko
V.E., Alimzhanov M.B., Turetskaya R.L., Ruhlmann A., Nordheim A., Kuprash D.V.,
Nedospasov S.A.
Molecular
Biology 35 (2001) 115-121
Membrane lymphotoxin (LT) is a heterotrimer LT12
, and its production depends on two genes. Northern blotting was employed in
studying their transcription in human B- and T-lymphoma cell lines and in
peripheral blood lymphocytes before and after induction with phorbol myristate
acetate (PMA). Transcription of either gene proved to be similarly regulated in
several cell lines and in blood lymphocytes. Activation of the LT gene was
associated with induction of transcription factor NF-B (p50/p65) upon cell
treatment with PMA. On evidence of RT–PCR, two transcripts of the LT gene were
present in equimolar amounts in all lymphoid cells. A product of alternative
splicing contained an open reading frame coding for the cytoplasmic portion of
LT.
Kuimov
A.N., Kuprash D.V., Petrov V.N., Vdovichenko K.K., Scanlan M.J., Jongeneel
C.V., Lagarkova M.A., Nedospasov S.A.
Genes and
Immunity 2 (2001) 52-55
By serological screening of a breast tumor cDNA
library we have identified a novel human gene, tnkl, encoding an
ankyrin-related protein with a high degree of similarity to tankyrase, the poly(ADP-ribose)polymerase
associated with human telomeres (Smith et al, Science 282: 1484). The tnkl gene
maps to chromosome 10, while the tnks gene encoding tankyrase is located on
chromosome 8. The predicted 1166-aa protein product of the tnkl gene is 78%
identical to human tankyrase and 62% to a putative D. melanogaster protein.
Since the proteins have essentially identical domain structures, the
corresponding genes form a distinct gene family. The possible link between TNKL
and cancer justifies its further functional analysis.
Bioenergetics and photosynthesis
Bakeeva
L.E., Skulachev V.P., Sudarikova Y.V., Tsyplenkova V.G.
Biochemistry
(Moscow) 66 (2001) 1335-1341
Electron
microscopy of cardiomyocytes of patients with hypertrophic and alcoholic
cardiomyopathies revealed the presence of nuclei with mitochondria accumulated
in their core. This was associated with chromatin displacement towards the core
of the nucleus. No large-scale intermixing of the nuclear content with the
cytosol was found, although in some sections there were disruptions in the
nuclear envelop continuity. The entrance of mitochondria into the nucleus was
modeled in rats that were given ethanol and the catalase inhibitor
aminotriazole for 12 weeks. It is suggested that the entrance of mitochondria
into the nucleus promotes both the attack of mitochondria by nuclear proteins
and the attack of nuclear DNA and proteins by proteins of the mitochondrial
intermembrane space.
Kalaidzidis
I.V., Kaulen A.D., Radionov A.N., Khitrina L.V.
Biochemistry
(Moscow) 66 (2001) 1220-1233
The scheme of the bacteriorhodopsin photocycle
associated with a transmembrane proton transfer and electrogenesis is
considered. The role of conformational changes in the polypeptide chain during
the proton transport is discussed.
Skulachev
V.P.
Biochemistry
(Moscow) 66 (2001) 1153-1156
This
paper considers the composition and function of sensory systems monitoring H2O2
level by the lung neuroepithelial cells and carotid bodies. These systems are
localized in the plasma membrane of the corresponding cells and are composed of
O2*--generating NADPH-oxidase and an H2O2-activated
K+ channel. This complex structure of the H2O2
sensors is probably due to their function in antioxidant defense. By means of
these sensors, an increase in the H2O2 level in lung or
blood results in a decrease in lung ventilation and constriction of blood
vessels. This action lowers the O2 flux to the tissues and, hence,
intracellular [O2]. The [O2] decrease, in turn, inhibits
intracellular generation of reactive oxygen species. The possible roles of such
systems under normal conditions (e.g., the effect of O2*- in air)
and in some pathologies (e.g., pneumonia) is discussed.
Noncoupled NADH:ubiquinone oxidoreductase of Azotobacter
vinelandii is required for diazotrophic growth at high oxygen
concentrations
Bertsova
Yu.V., Bogachev A.V., Skulachev V.P.
Journal
of Bacteriology 183 (2001) 6869-6874
The gene encoding the noncoupled NADH:ubiquinone
oxidoreductase (NDH II) from Azotobacter vinelandii was cloned,
sequenced, and used to construct an NDH II-deficient mutant strain. Compared to
the wild type, this strain showed a marked decrease in respiratory activity. It
was unable to grow diazotrophically at high aeration, while it was fully
capable of growth at low aeration or in the presence of NH4+.
This result suggests that the role of NDH II is as a vital component of the
respiratory protection mechanism of the nitrogenase complex in A. vinelandii.
It was also found that the oxidation of NADPH in the A. vinelandii respiratory
chain is catalyzed solely by NDH II.
Samartsev
V.N., Markova O.V., Chezghanova S.A., Mokhova E.N.
Biochemistry
(Moscow) 66 (2001) 926-931
The influence of the positively charged
amphiphilic compound cetyltrimethyl ammonium bromide (CTAB) on palmitate- and
laurate-induced uncoupling and on carboxyatractylate and glutamate recoupling
effects in liver mitochondria have been studied. CTAB (40 mM) in the presence of 3 mM MgCl2 had little (if any) effect on the
palmitic acid-stimulated respiration of mitochondria; the glutamate recoupling
effect increased, and the carboxyatractylate recoupling effect decreased to the
same degree with the combined effect (about 80%) remaining unchanged. Thus,
CTAB decreases the ADP/ATP antiporter involvement and increases to the same
extent the aspartate/glutamate antiporter involvement in the fatty acid-induced
uncoupling. The carboxyatractylate and glutamate recoupling effects were less
pH dependent in the presence of CTAB than in its absence. These data could be
interpreted with the assumption that fatty acid anions are more accessible to
the ADP/ATP antiporter and their neutral forms are more accessible to the
aspartate/glutamate antiporter, and that CTAB changes the relative anion
carrier involvement in the fatty acid-induced uncoupling as it forms neutral
complexes with fatty acid anions.
Rokitskaya
T.I., Zakharov S.D., Antonenko Yu.N., Kotova E.A., Cramer W.A.
FEBS
Letters 505 (2001) 147-150
The bacterial toxin colicin E1 is known to
induce voltage-gated currents across a planar bilayer lipid membrane. In the
present study, it is shown that the colicin-induced current decreased
substantially upon illumination of the membrane in the presence of the
photosensitizer, aluminum phthalocyanine. This effect was almost completely
abolished by the singlet oxygen quencher, sodium azide. Using single tryptophan
mutants of colicin E1, Trp495 was identified as the amino acid residue
responsible for the sensitized photodamage of the colicin channel activity.
Thus, the distinct participation of a specific amino acid residue in the
sensitized photoinactivation of a defined protein function was demonstrated. It
is suggested that Trp495 is critical for the translocation and/or anchoring of
the colicin channel domain in the membrane.
Vyssokikh
M.Y., Katz A., Rueck A., Wuensch C., Dorner A., Zorov D.B., Brdiczka D.
The
Biochemical Journal 358 (2001) 349-358
Different isoforms of the adenine nucleotide
translocase (ANT) are expressed in a tissue-specific manner. It was assumed
that ANT-1 and ANT-2 co-exist in every single mitochondrion and might be
differently distributed within the membrane structures that constitute the
peripheral inner membrane or the crista membrane. To discriminate between ANT
originating from peripheral or from cristal inner membranes we made use of the
fact that complexes between porin, the outer-membrane pore protein, and the ANT
can be generated. Such complexes between porin and the ANT in the peripheral
inner membrane were induced in rat heart mitochondria and isolated from rat
brain and kidney. Using ANT-isotype-specific antibodies and sequence analysis
of the N-terminal end, it was discovered that the peripheral inner membrane
contained ANT-1 and ANT-2, whereas the cristal membrane contained exclusively
ANT-2. Cyclophilin was co-purified with the porin-ANT complexes, whereas it was
absent in the crista-derived ANT. This suggested that ANT-1 might have a higher
affinity for cyclophilin. This specific intra-mitochondrial distribution of the
two ANT isotypes and cyclophilin D suggests specific functions of the
peripheral and crista-forming parts of the inner membrane and the two ANT
isotypes therein.
Mamedov
M.D., Mamedova A.A., Chamorovsky S.K., Semenov A.Yu.
FEBS
Letters 500 (2001) 172-176
An electrometric technique was used to
investigate electron transfer between spinach plastocyanin (Pc) and
photooxidized primary electron donor P700 in photosystem I (PS I) complexes
from the cyanobacterium Synechocystis sp. PCC 6803. In the presence of
Pc, the fast unresolvable kinetic phase of membrane potential generation
related to electron transfer between P700 and the terminal iron-sulfur acceptor
FB was followed by additional electrogenic phases in the microsecond
and millisecond time scales, which contribute approximately 20% to the overall
electrogenicity. These phases are attributed to the vectorial electron transfer
from Pc to the protein-embedded chlorophyll dimer P700+ within the
PsaA/PsaB heterodimer. The observed rate constant of the millisecond kinetic
phase exhibited a saturation profile at increasing Pc concentration, suggesting
the formation of a transient complex between Pc and PS I with the dissociation
constant Kd of about 80 mM. A small but detectable fast electrogenic
phase was observed at high Pc concentration. The rate constant of this phase
was independent of Pc concentration, indicating that it is related to a
first-order process.
Electrogenic proton transfer in Rhodobacter sphaeroides reaction
centers: effect of coenzyme Q10 substitution by decylubiquinone in
the QB binding site
Gopta
O.A., Semenov A.Yu., Bloch D.A.
FEBS
Letters 499 (2001) 116-120
An electrometric technique was used to
investigate the effect of coenzyme Q10 (UQ), substitution by
decylubiquinone (dQ) at the QB binding site of reaction centers
(UQ-RC and dQ-RC, respectively) on the electrogenic proton transfer kinetics
upon QB reduction in Rhodobacter sphaeroides chromatophores. Unlike
dQ-RC, the kinetics of the second flash-induced proton uptake in UQ-RC clearly
deviated from the mono-exponential one. The activation energy (about 30 kJ/mol)
and the pH profile of the kinetics in dQ-RC were similar to those in UQ-RC,
with the power law approximation used in the latter case. The interpretation of
the data presumed the quinone translocation between the two binding positions
within the QB site. It is proposed that the native isoprenyl side
chain (in contrast to decyl chain) favors the equilibrium binding of neutral
quinone at the redox-active 'proximal' position, but causes a higher barrier
for the hydroquinone movement from 'proximal' to 'distal' position.
Skulachev
V.P.
Experimental
Gerontology 36 (2001) 995-1024
Analysis of the programmed death phenomena from
mitochondria (mitoptosis) to whole organisms (phenoptosis) clearly shows that
suicide programs are inherent at various levels of organization of living
systems. Such programs perform very important functions, purifying (i) cells
from damaged (or unwanted for other reasons) organelles, (ii) tissues from
unwanted cells, (iii) organisms from organs transiently appearing during
ontogenesis, and (iv) communities of organisms from unwanted individuals.
Defence against reactive oxygen species (ROS) is probably one of primary
evolutionary functions of programmed death mechanisms. So far, it seems that
ROS play a key role in the mito-, apo-, organo- and phenoptoses. Here a concept
is described which tries to unite Weismann's concept of aging as an adaptive
programmed death mechanism and the alternative point of view considering aging
as an inevitable result of accumulation in an organism of occasional injuries.
It is suggested that injury accumulation is monitored by special system sending
a death signal to actuate a phenoptotic program when the number of injuries
reaches some critical level. The system in question is organized in such a way
that the lethal case appears to be a result of phenoptosis long before
occasional injuries make the functioning of the organism impossible. This
strategy is supposed to prevent the appearance of asocial monsters capable to
ruining kin, community and entire population. These relationships are regarded
as an example of the Samurai law of biology: 'It is better to die than to be
wrong'. It is stressed that for humans these cruel regulations look like an
atavism that should be overcome to prolong the human life span.
Barbara
Cannon's data on the UCP1-ablated mice: "non-cannonical" point of
view.
Skulachev
V.P.
Bioscience
Reports 21 (2001) 189-194
The data of Cannon and co-workers on
UCP1-ablated mice are interpreted assuming that UCP2 and UCP3 are involved in
thermoregulation as fatty acid-dependent uncouplers although they are not
sufficient, in the absence of UCP1, for long term maintenance of normal body
temperature of mice after sudden and strong decrease in the ambient
temperature. I would like to suggest that in brown fat of control mice, UCP1 is
present in an amount higher than UCP2 and 3 and, therefore, is able to cause
(a) some fatty acid-mediated decrease in proton motive force in resting state
and, hence, (b) oxidation of CoQH2 to CoQ which is shown by Klingenberg and
coworkers to be cofactor for UCPs. This results in strong uncoupling and
thermogenesis mediated by UCP1, 2 and 3. In the UCP1-ablated mice, activity of
UCP2 and 3 appears to be insufficient to induce CoQH2 oxidation in resting
brown fat mitochondria, which results in hypothermia.
Bogachev
A.V., Bertsova Yu.V., Barquera B., Verkhovsky M.I.
Biochemistry
40 (2001) 7318-7323
The Na+-translocating NADH:ubiquinone
oxidoreductase (Na+-NQR) from Vibrio harveyi was purified and
studied by EPR and visible spectroscopy. Two EPR signals in the NADH-reduced
enzyme were detected: one, a radical signal, and the other a line around g =
1.94, which is typical for a [2Fe-2S] cluster. An Em of -267 mV was
found for the Fe-S cluster (n = 1), independent of sodium concentration. The
spin concentration of the radical in the enzyme was approximately the same
under a variety of redox conditions. The time course of Na+-NQR
reduction by NADH indicated the presence of at least two different flavin
species. Reduction of the first species (most likely, a FAD near the NADH
dehydrogenase site) was very rapid in both the presence and absence of sodium.
Reduction of the second flavin species (presumably, covalently bound FMN) was
slower and strongly dependent on sodium concentration, with an apparent
activation constant for Na+ of approximately 3.4 mM. This is very
similar to the Km for Na+ in the steady-state quinone reductase
reaction catalyzed by this enzyme. These data led us to conclude that the
sodium-dependent step within the Na+-NQR is located between the
noncovalently bound FAD and the covalently bound FMN.
Borisov
A.Yu.
Membrane
& Cell Biology 14 (2000) 333-341
According to the current model of primary events
in plants, electronic excitations generated in antenna chlorophylls (Chl) by
the light rapidly migrate within vast Chl ensembles, reach the reaction centres
(RCs) and initiate the primary photoreactions of electron transfer from the RC
special pairs (P700 and P680 in plant photosystems). A minor portion of
electronic excitations is lost en route, in particular, via fluorescence of Chl
a. A number of fluorescence parameters in vivo had been reliably
established in many independent studies. Based on these parameters, the author
calculated a dimensionless value, the ratio of fluorescence yields emitted by
PS-2 and PS-1 Chl a ensembles. The ratio proved to differ 5-10 times from that
obtained in experiments. Such a divergence seems to indicate an internal
discrepancy in the currently used model. The author proposes a substantial
modernization of the model by introducing a new subpicosecond state for RCs,
which precedes the primary reaction of electron transfer from the RC special
pairs.
Simonyan
R.A., Jimenez M., Ceddia R.B., Giacobino J.P., Muzzin P., Skulachev V.P.
Biochimica
et Biophysica Acta 1505 (2001) 271-279
The mechanism of thermoregulatory uncoupling of
respiration and phosphorylation in skeletal muscles has been studied. It is
found that 24 h cold exposure results in (i) a 3-fold increase in the amount of
UCP3 protein in rat skeletal muscle mitochondria, and (ii) pronounced lowering
of the membrane potential in isolated rat or mouse skeletal muscle
mitochondria. The decrease in membrane potential is reversed by adding bovine
serum albumin. Cold exposure is also found to sensitize the membrane potential
to the uncoupling action of added fatty acid (laurate). After laurate addition,
the recoupling effects of GDP and carboxyatractylate decrease whereas that of albumin
increases in mitochondria from cold-treated rats or mice. Changes similar to
those induced by cold can be initiated by the in vivo addition of
thyroxine. Cold exposure does not affect energy coupling in liver mitochondria.
The possible involvement of UCP3 isoforms in nucleotide-sensitive and
-insensitive uncoupling is discussed.
Borisov
V.B., Sedelnikova S.E., Poole R.K., Konstantinov A.A.
The
Journal of Biological Chemistry 276 (2001) 22095-22099
Azotobacter vinelandii is an obligately aerobic bacterium in which
aerotolerant dinitrogen fixation requires cytochrome bd. This oxidase comprises
two polypeptide subunits and three hemes, but no copper, and has been studied
extensively. However, there remain apparently conflicting reports on the
reactivity of the high spin heme b595 with ligands. Using
purified cytochrome bd, we show that absorption changes induced by CO
photodissociation from the fully reduced cytochrome bd at low
temperatures demonstrate binding of the ligand with heme b595.
However, the magnitude of these changes corresponds to the reaction with CO of
only about 5% of the heme. CO binding with a minor fraction of heme b595
is also revealed at room temperature by time-resolved studies of CO
recombination. The data resolve the apparent discrepancies between conclusions
drawn from room and low temperature spectroscopic studies of the CO reaction
with cytochrome bd. The results are consistent with the proposal that
hemes b595 and d form a diheme oxygen-reducing center with a
binding capacity for a single exogenous ligand molecule that partitions between
the hemes d and b595 in accordance with their
intrinsic affinities for the ligand. In this model, the affinity of heme b595
for CO is about 20-fold lower than that of heme d.
Yakovlev
A.G., Shuvalov V.A.
Biochemistry
(Moscow) 66 (2001) 211-220
The process of electron transfer from the
primary electron donor P* to the primary electron acceptor BA in the
reaction center of Rhodobacter sphaeroides R-26 under 30 fsec pulse
excitation was studied in this work with the aim of establishing a relationship
between the nuclear subsystem motion and charge transfer. For this purpose the
fsec and psec oscillations in the bands of stimulated emission of P* and in the
band of reaction product BA- at 1020 nm were investigated. It was
established that the reversible formation of the P+BA-
state is characterized by two vibration modes (130 and 320 cm-1) and
connected with an arrival of the wavepacket induced by fsec excitation to the
intersection of potential surfaces P*BA and P+BA-.
The irreversible formation of the P+BA- state
with the time constant of 3 psec is followed by oscillations with frequencies
of 9 and 33 cm-1. These results show that the irreversibility of
electron transfer is determined by two factors: 1) by a difference between the
energy width of the wavepacket and the gap between the named surfaces; 2) by a
difference between the duration of wavepacket residence near the intersection
of the surfaces and the relaxation time of the P+BA-
state.
Skulachev
V.P.
FEBS
Letters 492 (2001) 1-3
Skulachev
V.P.
Biochemistry
(Moscow) 65 (2000) 749-750
Borisov
A.Yu.
Biochemistry
(Moscow) 65 (2000) 1429-1434
Although the two electron-transfer branches in
the reaction centers (RC) of purple bacteria are virtually symmetric, it is
well known that only one of them is functionally active (the A-branch). The
mechanisms of functional asymmetry of structurally symmetric branches of the
electron transport system are analyzed in this work within the framework of the
theory of bimolecular charge-transfer complexes (CTC). CTC theory is shown to
provide an explanation of this phenomenon. According to the CTC theory, the
dominance of one branch is required to implement the CTC state in special
bacteriochlorophyll pairs of RC, in which more than 30% of the excited electron
density in the CTC is shifted toward one of the bacteriochlorophyll molecules.
This causes a significant increase in the efficiency of further electron transfer
to the primary quinone acceptor as compared to a system with two absolutely
symmetric electron transfer branches. Specific features of dielectric asymmetry
near the RC special pair are discussed. It is emphasized that a strong CTC is
able to provide effective trapping of electronic excitation energy from antenna
chlorophyll, which is a main function of the RC. Hypothetical stages of CTC
formation in other classes of photosynthesizing bacteria during evolution are
discussed.
Skulachev
V.P.
Trends in
Biochemical Sciences 26 (2001) 23-29
Mitochondria exist in two interconverting forms;
as small isolated particles, and as extended filaments, networks or clusters
connected with intermitochondrial junctions. Extended mitochondria can
represent electrically united systems, which can facilitate energy delivery
from the cell periphery to the cell core and organize antioxidant defence of
the cell interior when O2 is consumed by mitochondrial clusters near
the the outer cell membrane, and protonic potential is transmitted to the cell
core mitochondria to form ATP. As to small mitochondria, they might represent a
transportable form of these organelles.
van
Grondelle R., Novoderezhkin V.
Biochemistry
40 (2001) 15057-15068
Photosynthetic
light harvesting is a unique life process that occurs with amazing efficiency.
Since the discovery of the structure of the bacterial peripheral
light-harvesting complex (LH2), this process has been studied using a variety
of advanced laser spectroscopic methods. We are now in a position to discuss
the physical origins of excitation energy transfer and trapping in the LH2 and
LH1 antennae of photosynthetic purple bacteria. We demonstrate that the time
evolution of the state created by the light is determined by the combined
action of excitonic pigment-pitment interactions, energetic disorder, and
coupling to nuclear motion in a pigment-protein complex. A quantitative fit of
experimental data using Redfield theory allowed us to determine the pathways
and time scales of exciton and vibrational relaxation and analyze separately
different contributions to the measured transient absorption dynamics.
Furthermore, these dynamics were observed to be strongly dependent on the
excitation wavelength. A numerical fit of this dependence turns out to be
extremely critical to a variation of the structure and disorder parameters and,
therefore, can be used as a test for different antenna models (disordered ring,
elliptical deformations, correlated disorder, etc.). The calculated
equilibration dynamics in the exciton basis allow a visualization of the
exciton motion using a density matrix picture in real space.
Unit building block of the oligomeric chlorosomal antenna of the
green photosynthetic bacterium Chloroflexus aurantiacus: modeling of nonlinear
optical spectra
Novoderezhkin
V., Taisova A., Fetisova Z.
Chemical
Physics Letters 335 (2001) 234-240
The size of a unit BChl c aggregate of the
chlorosomal antenna of the green photosynthetic bacterium Chloroflexus
aurantiacus was estimated by using the relative difference absorption
measurements of the oligomeric BChl c and monomeric BChl a bands [S. Savikhin
et al., FEBS Lett. 430 (1998) 323]. A quantitative fit of the data was obtained
within the framework of the exciton model proposed before [Z.G. Fetisova et
al., Biophys. J. 71 (1996) 995]. The model assumes that a unit building block
of the antenna has a form of a tubular aggregate of L = 6 linear chains,
containing N BChl c molecules each. The simultaneous fit of the linear and
nonlinear (pump-probe) absorption gave the intrachain interaction energy of 400
cm-1, the site inhomogeneity value of 280 cm-1, and the
number of pigments in each individual linear chain N = 4. The size of the
antenna unit was found to be L x N = 24 exciton-coupled BChl c molecules.
Borisov
A.Yu.
Biochemistry
(Moscow) 65 (2000) 1266-1271
Using computer simulation, it was found that, at
least in case of purple bacteria, the widely accepted model for the primary
photosynthetic processes (energy migration to reaction centers and its further
trapping) is unable to provide a fully consistent explanation for the available
experimental data. Two physical mechanisms suggested in this work are thought
to be able to resolve or at least decrease the severity of this problem.
Shapovalov
V.L., Rokitskaya T.I., Kotova E.A., Krokhin O.V., Antonenko Y.N.
Photochemistry
and Photobiology 74 (2001) 1-7
Interaction of potent photodynamic agents,
sulfonated aluminum phthalocyanines (AlPcSn where n is a number of sulfonic
groups), with biological membranes was studied here using model systems:
sensitized photoinactivation of gramicidin channels in planar lipid bilayers
and adsorption on lipid monolayers. Fluoride anions known to form complexes
with aluminum were found to inhibit both the adsorption of aluminum
phthalocyanines on lipid monolayers, as measured with a Langmuir trough by
surface pressure and surface potential changes, and photodynamic efficacy of
the dyes, as studied by gramicidin channel photoinactivation. The similar
effects were caused by the alkalinization of the medium. Fluoride anions
appeared to be much more effective in the case of AlPcS4 as compared to AlPcS3.
The suppression of the photodynamic potency of aluminum phthalocyanines was
attributed to desorption of the dyes from lipid bilayers induced by fluoride or
hydroxyl ions. With AlPcS4 an enhancement of the dye aggregation leading to a
decrease in the sensitizing activity was probably involved in the fluoride
effect as revealed by absorption and fluorescence spectral measurements.
Capillary electrophoresis was employed to understand the mechanism of the dye
desorption. The results of these experiments indicated that the reduction in
the membrane affinity was associated with an increase in the negative charge of
the dye molecules due to the binding of fluoride or hydroxyl ions.
Bodrova
M.E., Dedukhova V.I., Samartsev V.N., Mokhova E.N.
IUBMB
Life 50 (2000) 189-194
We show that Ca2+ loading of
mitochondria substantially augments the myristate-induced decrease in the
transmembrane electric potential difference DY. Such a Ca2+ action is without effect on the
respiration rate and is not accompanied by the high-amplitude swelling when low
concentrations of Ca2+ and myristate are used. The myristate-inducedDY decrease is prevented and reversed by
cyclosporin A (CsA); the decrease is prevented and transiently reversed by
nigericin. To explain these effects, we suggest that myristate induces opening
of the mitochondrial permeability transition pore at a low-conductance state.
Addition of carboxyatractylate (CAtr) after myristate induces the CsA-sensitive
uncoupling, but when added after myristate and CsA, CAtr produces a decrease inDY, if the interval between myristate and CsA
addition is sufficiently long. The CAtr effect is completely reversed by EGTA
and transiently reversed by nigericin. This suggests that the
ADP/ATP-antiporter participates in the CsA-sensitive uncoupling when present as
a pore complex constituent. ADP/ATP-antiporter that does not take part in the
pore complex formation is involved in the CsA-insensitive uncoupling.
Brailovskaya I.V., Starkov
A.A., Mokhova E.N.
Biochemistry (Moscow) 66
(2001) 909-912
Oxidative
stress is one of the most frequent causes of tissue and cell injury in various
pathologies. The molecular mechanism of mitochondrial damage under conditions
of oxidative stress induced in vitro with low concentrations of FeSO4
and ascorbate (vitamin C) was studied. FeSO4 (1-4 mM) added to rat liver mitochondria
that were incubated in the presence of 2.3 mM ascorbate induced (with a certain
delay) a decrease in membrane potential and high-amplitude swelling. It also
significantly decreased the ability of mitochondria to accumulate exogenous Ca2+.
All the effects of FeSO4 + ascorbate were essentially prevented by
cyclosporin A, a specific inhibitor of the mitochondrial Ca2+-dependent
pore (also known as the mitochondrial permeability transition). EGTA restored
the membrane potential of mitochondria de-energized with FeSO4 +
ascorbate. We hypothesize that oxidative stress induced in vitro with
FeSO4 and millimolar concentrations of ascorbate damages
mitochondria by inducing the cyclosporin A-sensitive Ca2+-dependent
pore in the inner mitochondrial membrane.
Lithoautotrophic growth of the freshwater strain
Grabovich M.Y., Patritskaya
V.Y., Muntyan M.S., Dubinina G.A.
FEMS Microbiology Letters
204 (2001) 341-345
The freshwater filamentous
bacterium Beggiatoa D-402 was shown to grow lithoautotrophically in a
homogeneous culture under microoxic conditions only, the growth yield being the
highest at 0.1 mg O2 l-1. High activities of the Calvin
cycle key enzymes and of the dissimilatory path thiosulfate oxidation enzymes
were found in the bacterial cells. The rate of CO2 fixation above 112
nmol min-1 (mg protein)-1, an about 90% increase in the
protein carbon at the expense of CO2 carbon and an increase in the
molar yield up to 12 mg dry weight (mmol oxidized thiosulfate)-1
indicate the bacterial growth was autotrophic. Thiosulfate was oxidized by the
strain almost completely into sulfate. The metabolically useful energy was
conserved by oxidative phosphorylation that was coupled to oxidation of sulfur
compounds. The bacterial membranes were found to contain CO-binding cytochromes
b and two cytochromes c with Mr 23 and 26 kDa, the terminal part of
the respiratory chain containing presumably a cbb3-type oxidase. A
cytochrome c with Mr 12 kDa was detected in the soluble fraction.
Oligomeric C-terminal truncated Bax preferentially releases
cytochrome c but not adenylate kinase from mitochondria, outer membrane
vesicles and proteoliposomes
Wieckowski M.R., Vyssokikh M.,
Dymkowska D., Antonsson B., Brdiczka D., Wojtczak L.
FEBS Letters 505 (2001)
453-459
The
mechanism by which the proapoptotic protein Bax releases cytochrome c from
mitochondria is not fully understood. The present work approaches this problem
using C-terminal truncated oligomeric Bax (BaxDeltaC). Micromolar
concentrations of BaxDeltaC released cytochrome c from isolated rat heart and
liver mitochondria, while the release of adenylate kinase was not significantly
affected. BaxDeltaC also released cytochrome c but not adenylate kinase from
outer membrane vesicles filled with these proteins. However, BaxDeltaC was
ineffective in releasing cytochrome c when outer membrane vesicles were
obtained in the presence of glycerol, conditions under which the number of
contact sites was drastically reduced. BaxDeltaC did not liberate encapsulated
cytochrome c and adenylate kinase from pure phospholipid vesicles or vesicles
reconstituted with porin. However, when the hexokinase-porin-adenine nucleotide
translocase complex from brain mitochondria was reconstituted in vesicles,
BaxDeltaC released internal cytochrome c but not adenylate kinase. In all these
systems, only a small portion of total cytochrome c present in either
mitochondria or vesicles could be liberated by BaxDeltaC. BaxDeltaC also
increased the accessibility of external cytochrome c to either oxidation by
complex IV or reduction by complex III in intact liver and heart mitochondria.
CONCLUSIONS: (1) BaxDeltaC selectively releases cytochrome c and enables a
bidirectional movement of cytochrome c across the outer mitochondrial membrane.
(2) A multiprotein complex that resembles the mitochondrial contact sites is a
prerequisite for BaxDeltaC action. (3) A limited pool of cytochrome c becomes
the first target for BaxDeltaC.
Cherepanov D.A., Krishtalik
L.I., Mulkidjanian A.Y.
Biophysical Journal 80
(2001) 1033-1049
Relaxation
processes in proteins range in time from picoseconds to seconds.
Correspondingly, biological electron transfer (ET) could be controlled by slow
protein relaxation. We used the Langevin stochastic approach to describe this
type of ET dynamics. Two different types of kinetic behavior were revealed,
namely: oscillating ET (that could occur at picoseconds) and monotonically
relaxing ET. On a longer time scale, the ET dynamics can include two different
kinetic components. The faster one reflects the initial, nonadiabatic ET,
whereas the slower one is governed by the medium relaxation. We derived a
simple relation between the relative extents of these components, the change in
the free energy (DG), and the energy of the slow reorganization L. The rate of ET was found to be
determined by slow relaxation at -DG < or = L. The application of the developed
approach to experimental data on ET in the bacterial photosynthetic reaction
centers allowed a quantitative description of the oscillating features in the
primary charge separation and yielded values of L for the slower low-exothermic ET
reactions. In all cases but one, the obtained estimates of L varied in the range of 70-100 meV.
Because the vast majority of the biological ET reactions are only slightly
exothermic (DG > or = -100 meV), the relaxationally controlled
ET is likely to prevail in proteins.
Ahlbrink R., Semin B.K.,
Mulkidjanian A.Y., Junge W.
Biochimica
et Biophysica Acta 1506 (2001) 117-126
The
catalytic Mn cluster of the photosynthetic oxygen-evolving system is oxidized
via a tyrosine, Y(Z), by a photooxidized chlorophyll a moiety, P+680.
The rapid reduction of P+680 by Y(Z) in nanoseconds
requires the intactness of an acid/base cluster around Y(Z) with an apparent
functional pK of <5. The removal of Mn (together with bound Ca) shifts the
pK of the acid/base cluster from the acid into the neutral pH range. At
alkaline pH the electron transfer (ET) from Y(Z) to P+680
is still rapid (<1 ms), whereas at acid pH the ET is much slower
(10-100 ms) and steered by proton release.
In the intermediate pH domain one observes a mix of these kinetic components
(see R. Ahlbrink, M. Haumann, D. Cherepanov, O. Bogershausen, A. Mulkidjanian,
W. Junge, Biochemistry 37 (1998)). The overall kinetics of P+680
reduction by Y(Z) in Mn-depleted photosystem II (PS II) has been previously
shown to be slowed down by divalent cations (added at >10 mM), namely: Mn2+, Co2+,
Ni2+, Cu2+, Zn2+ (C.W. Hoganson, P.A. Casey,
O. Hansson, Biochim. Biophys. Acta 1057 (1991)). Using Mn-depleted PS II core
particles from pea as starting material, we re-investigated this phenomenon at
nanosecond resolution, aiming at the effect of divalent cations on the
particular kinetic components of P+680 reduction. To our
surprise we found only the slower, proton steered component retarded by some
added cations (namely Co2+/Zn2+>Fe2+>Mn2+).
Neither the fast component nor the apparent pK of the acid/base cluster around
Y(Z) was affected. Apparently, the divalent cations acted (electrostatically)
on the proton release channel that connects the oxygen-evolving complex with
the bulk water, but not on the ET between Y(Z) and P+680,
proper. Contrastingly, Ca2+ and Mg2+, when added at >5
mM, accelerated the slow component of P+680 reduction by
Y(Z) and shifted the apparent pK of Y(Z) from 7.4 to 6.6 and 6.7, respectively.
It was evident that the binding site(s) for added Ca2+ and Mg2+
were close to Y(Z) proper. The data obtained are discussed in relation to the
nature of the metal-binding sites in photosystem II.
van Rotterdam B.J.,
Westerhoff H.V., Visschers R.W., Bloch D.A., Hellingwerf K.J., Jones M.R.,
Crielaard W.
European
Journal of Biochemistry / FEBS 268 (2001) 958-970
Transduction
of free-energy by Rhodobacter sphaeroides
reaction-center-light-harvesting-complex-1 (RC-LH1) was quantified. RC-LH1
complexes were reconstituted into liposomal membranes. The capacity of the
RC-LH1 complex to build up a proton motive force was examined at a range of
incident light intensities, and induced proton permeabilities, in the presence
of artificial electron donors and acceptors. Experiments were also performed
with RC-LH1 complexes in which the midpoint potential of the reaction center
primary donor was modified over an 85-mV range by replacement of the tyrosine
residue at the M210 position of the reaction center protein by histidine,
phenylalanine, leucine or tryptophan. The intrinsic driving force with which
the reaction center pumped protons tended to decrease as the midpoint potential
of the primary donor was increased. This observation is discussed in terms of
the control of the energetics of the first steps in light-driven electron
transfer on the thermodynamic efficiency of the bacterial photosynthetic
process. The light intensity at which half of the maximal proton motive force
was generated, increased with increasing proton permeability of the membrane.
This presents the first direct evidence for so-called backpressure control
exerted by the proton motive force on steady-state cyclic electron transfer
through and coupled proton pumping by the bacterial reaction center.
Pecoraro C., Gennis R.B.,
Vygodina T.V., Konstantinov A.A.
Biochemistry 40 (2001)
9695-9708
The
reaction of cytochrome c oxidase (COX) from Rhodobacter sphaeroides with
hydrogen peroxide has been studied at alkaline (pH 8.5) and acidic (pH 6.5)
conditions with the aid of a stopped-flow apparatus. Absorption changes in the
entire 350-800 nm spectral range were monitored and analyzed by a global
fitting procedure. The reaction can be described by the sequential formation of
two intermediates analogous to compounds I and II of peroxidases: oxidized COX
+ H2O2 --> intermediate I --> intermediate II. At
pH as high as 8.5, intermediate I appears to be a mixture of at least two
species characterized by absorption bands at approximately 607 nm (P607)
and approximately 580 nm (F-I580) that rise synchronously. At acidic
pH (6.5), intermediate I is represented mainly by a component with an a-peak around 575 nm (F-I575)
that is probably equivalent to the so-called F species observed with the bovine
COX. The data are consistent with a pH-dependent reaction branching at the step
of intermediate I formation. To get further insight into the mechanism of the
pH-dependence, the peroxide reaction was studied using two mutants of the R.
sphaeroides oxidase, K362M and D132N, that block, respectively, the
proton-conducting K- and D-channels. The D132N mutation does not affect
significantly the Ox --> intermediate I step of the peroxide reaction. In
contrast, K362M replacement exerts a dramatic effect, eliminating the
pH-dependence of intermediate I formation. The data obtained allow us to
propose that formation of the acidic form of intermediate I (F-I575,
F) requires protonation of some group at/near the binuclear site that follows
or is concerted with peroxide binding. The protonation involves specifically
the K-channel. Presumably, a proton vacancy can be generated in the site as a
consequence of the proton-assisted heterolytic scission of the O-O bond of the
bound peroxide. The results are consistent with a proposal [Vygodina, T. V.,
Pecoraro, C., Mitchell, D., Gennis, R., and Konstantinov, A. A. (1998)
Biochemistry 37, 3053-3061] that the K-channel may be involved in the delivery
of the first four protons in the catalytic cycle (starting from reduction of
the oxidized form) including proton uptake coupled to reduction of the
binuclear site and transfer of protons driven by cleavage of the dioxygen O-O
bond in the binculear site. Once peroxide intermediate I has been formed,
generation of a strong oxene ligand at the heme a3 iron triggers a transition of the
enzyme to the "peroxidase conformation" in which the K-channel is
closed and the binuclear site becomes protonically disconnected from the bulk
aqueous phase.
Zhang J., Hellwig P., Osborne
J.P., Huang H.W., Moenne-Loccoz P., Konstantinov A.A., Gennis R.B.
Biochemistry 40 (2001)
8548-8556
Cytochrome bd is
one of the two quinol oxidases in the respiratory chain of Escherichia coli.
The enzyme contains three heme prosthetic groups. The dioxygen binding site is
heme d, which is thought to be part of the heme-heme binuclear center along
with heme b595, which is a high-spin heme whose function is
not known. Protein sequence alignments [Osborne, J. P., and Gennis, R. B.
(1999) Biochim. Biophys Acta 1410, 32--50] of cytochrome bd quinol
oxidase sequences from different microorganisms have revealed a highly
conserved sequence (GWXXXEXGRQPW; bold letters indicate strictly conserved
residues) predicted to be on the periplasmic side of the membrane between
transmembrane helices 8 and 9 in subunit I. The functional importance of this
region is investigated in the current work by site-directed mutagenesis.
Several mutations in this region (W441A, E445A/Q, R448A, Q449A, and W451A)
resulted in a catalytically inactive enzyme with abnormal UV-vis spectra. E445A was selected for
detailed analysis because of the absence of the absorption bands from heme b595.
Detailed spectroscopic and chemical analyses, indeed, show that one of the
three heme prosthetic groups in the enzyme, heme b595, is
specifically perturbed and mostly missing from this mutant. Surprisingly, heme d, while known
to interact with heme b(595), appears relatively unperturbed, whereas the
low-spin heme b(558) shows some modification. This is the first report of a
mutation that specifically affects the binding site of heme b(595).
Mironova E.V., Lukin A.Y.,
Shevyakov S.V., Alexeeva S.G., Shvets V.I., Demina O.V., Khodonov A.A.,
Khitrina L.V.
Biochemistry (Moscow) 66
(2001) 1323-1335
A
method for synthesis of retinal analogs labeled with electron-density groups is
suggested. The interaction of these polyene compounds with bacterioopsin in
apomembrane of Halobacterium salinarum was tested. A retinal analog containing
a crown-ether receptor group is able to interact readily with bacterioopsin
giving rise to rapid formation of a pigment with absorption maximum at 460 nm.
This pigment is capable of undergoing cyclic photoconversion. The
crown-bacteriorhodopsin photocycle is extremely slow and its quantum efficiency
is very low (~3% of that in native bacteriorhodopsin). This photocycle includes
an M-like intermediate with a differential absorption maximum at 380 nm. A
retinal analog in which the b-ionone ring is replaced by ferrocene moiety forms a
stable chromoprotein with the main absorption band at 483 nm and a shoulder
near 590-610 nm.
Cherepanov
D.A., Mulkidjanian A.Y.
Biochimica
et Biophysica Acta 1505 (2001) 179-184
In ferredoxin I from Azotobacter vinelandii,
the reduction of a [3Fe-4S] iron-sulphur cluster is coupled with the
protonation of the mu2S sulphur atom that is approx. 6 A away from the protein
boundary. The recent study of the site-specific mutants of ferredoxin I led to
the conclusion that a particular surface aspartic residue (Asp15) is solely
responsible for the proton transfer to the mu2S atom by 'rapid penetrative
excursions' (K. Chen, J. Hirst, R. Camba, C.A. Bonagura, C.D. Stout, B.K.
Burgess, F.A. Armstrong, Nature 405 (2000) 814-817). In the same paper it has
been reported that the replacement of Asp15 by glutamate led to the blockage of
the enzyme, although glutamate, with its longer and more flexible side chain,
should apparently do even better as a mobile proton carrier than aspartate. We
tackled this puzzling incompetence of Glu15 by molecular dynamics simulations.
It was revealed that the conformational alterations of Asp15 are energetically
balanced by the straining of the nearby Lys84 side chain in wild-type
ferredoxin I but not in the Asp15-->Glu mutant. Lys84 in ferredoxin I of A.
vinelandii seems to represent the first case where the strained (entatic)
conformation of a particular amino acid side chain could be directly identified
in the ground state of an enzyme and assigned to a distinct mechanism of energy
balance during the catalytic transition.
Kalinina N.O.,
Rakitina D.A., Yelina N.E., Zamyatnin A.A. Jr, Stroganova T.A., Klinov D.V.,
Prokhorov V.V., Ustinova S.V., Chernov B.K., Schiemann J., Solovyev A.G.,
Morozov S.Y.
Journal
of General Virology 82 (2001) 2569-2578
The 63 kDa '63K' movement protein encoded by the
triple gene block of poa semilatent virus (PSLV) comprises the C-terminal
NTPase/helicase domain and the N-terminal extension domain, which contains two
positively charged sequence motifs, A and B. In this study, the in vitro
RNA-binding properties of PSLV 63K and its mutants were analysed.
Membrane-immobilized 63K and N-63K (isolated N-terminal extension domain) bound
RNA at high NaCl concentrations. In contrast, C-63K (isolated NTPase/helicase
domain) was able to bind RNA only at NaCl concentrations of up to 50 mM. In
gel-shift assays, C-63K bound RNA to form complexes that were unable to enter
an agarose gel, whereas complexes formed by N-63K could enter the gel.
Full-length 63K formed both types of complexes. Visualization of the RNA-protein
complexes formed by 63K, N-63K and C-63K by atomic force microscopy
demonstrated that each complex had a different shape. Collectively, these data
indicate that 63K has two distinct RNA-binding activities associated with the
NTPase/helicase domain and the N-terminal extension domain. Mutations in either
of the positively charged sequence motifs A and B had little effect on the RNA
binding of the N-terminal extension domain, whereas mutations in both motifs
together inhibited RNA binding. Hybrid viruses with mutations in motifs A and B
were able to infect inoculated leaves of Nicotiana benthamiana plants, but were
unable to move systemically to uninoculated leaves, suggesting that the
RNA-binding activity of the N-terminal extension domain of PSLV 63K is associated
with virus long-distance movement.
Baratova
L.A., Efimov A.V., Dobrov E.N., Fedorova N.V., Hunt R., Badun G.A., Ksenofontov
A.L., Torrance L., Jarvekulg L.
Journal
of Virology 75 (2001) 9696-9702
Potato virus A (PVA) particles were bombarded
with thermally activated tritium atoms, and the intramolecular distribution of
the label in the amino acids of the coat protein was determined to assess their
in situ steric accessibility. This method revealed that the N-terminal
15 amino acids of the PVA coat protein and a region comprising amino acids 27
to 50 are the most accessible at the particle surface to labeling with tritium
atoms. A model of the spatial arrangement of the PVA coat protein polypeptide
chain within the virus particle was derived from the experimental data obtained
by tritium bombardment combined with predictions of secondary-structure
elements and the principles of packing a-helices and b-structures in proteins. The model predicts three regions of
tertiary structure: (i) the surface-exposed N-terminal region, comprising an
unstructured N terminus of 8 amino acids and two b-strands, (ii) a C-terminal region including
two a-helices, as well as three b-strands that form a two-layer structure called
an abCd unit, and (iii) a central region comprising a bundle of four a-helices in a fold similar to that found in
tobacco mosaic virus coat protein. This is the first model of the three-dimensional
structure of a potyvirus coat protein.
Bazhin
A.V., Shifrina O.N., Savchenko M.S., Tikhomirova N.K., Goncharskaia M.A.,
Gorbunova V.A., Senin I.I., Chuchalin A.G., Philippov P.P.
Lung
Cancer 34 (2001) 99-104
To date, many authors have described the
presence of autoantibodies against various neuronal proteins, paraneoplastic
antigens (PNA), in a serum of patients with different kinds of malignant tumors
located outside the nervous system. These autoantibodies may cross-react with
the corresponding PNA or their epitopes present in neurons and thus initiate
the development of a variety of neurological disorders, paraneoplastic syndromes
(PNS), even though the primary tumor and its metastases have not invaded the
nervous system. Cancer-associated retinopathy (CAR) is a rare ocular PNS
induced by autoantibodies against several retinal antigens, one of which is a
photoreceptor calcium-binding protein, recoverin. Only several CAR patients
with a few kinds of cancer (endothelial carcinoma, breast cancer, epithelial
ovarian carcinoma) have so far been found to contain autoantibodies against
recoverin in their sera. As for lung cancer, the majority of CAR cases mediated
by anti-recoverin autoantibodies have been revealed in patients with the most
malignant lung cancer, small cell lung carcinoma (SCLC), and only one similar
case has been described for a patient with non-small lung carcinoma. The common
feature of all these anti-recoverin-positive patients, irrespective of the type
of cancer, is the presence of both the CAR syndrome and high titres (as a rule,
more than 1:1000) of the underlying autoantibodies in their serum. In this
study, we have used recombinant myristoylated recoverin to screen serum samples
of 50 patients with SCLC by Western blot and revealed 5 individuals with low
titres of anti-recoverin antibodies, who have no manifestation of a loss of
vision. To our knowledge, this is the first report on the presence of low titre
autoantibodies against recoverin in a serum of patients with cancer, but
without visual dysfunction.
Atabekov
J.G., Rodionova N.P., Karpova O.V., Kozlovsky S.V., Novikov V.K., Arkhipenko
M.V.
Virology
286 (2001) 466-474
Previously we showed that encapsidated potato
virus X (PVX) RNA is nontranslatable in vitro, but can be converted into
a translatable form after binding to PVX particles of PVX-coded movement
protein, the product of the first gene of triple gene block (TGBp1). Here we
report that a similar effect occurs via in situ phosphorylation of the
PVX coat protein (CP) by Ser/Thr protein kinase (PK) C, the mixture of casein
kinases I and II or by cytoplasmic PKs from Nicotiana glutinosa
leaves. Immunochemical analyses indicated that phosphorylation induced
conformational changes in PVX CP. The N-terminal region of the PVX CP, rich in
Ser and Thr residues, is exposed at the virion surface and can be removed by
treatment with trypsin. We showed that (i) trypsin treatment removed the bulk
of 32P-radioactivity from in situ phosphorylated PVX CP, (ii)
PVX containing N-terminally truncated CP (PVX-Ptd) failed to be translationally
activated by phosphorylation, and (iii) the specific infectivity of PVX-Ptd was
reduced. However, the PVX-Ptd RNA remained intact and PVX-Ptd could be
translationally activated by the PVX MP TGBp1. We hypothesize that phosphorylation
of the parental PVX by cytoplasmic PKs in vivo renders PVX
RNA translatable in primary inoculated cells, whereas translational activation
of the progeny virions destined for plasmodesmata trafficking is triggered by
TGBp1.
Kiselyova
O.I., Yaminsky I.V., Karger E.M., Frolova O.Y., Dorokhov Y.L., Atabekov J.G.
Journal
of General Virology 82 (2001) 1503-1508
The structure of complexes formed in vitro by
tobacco mosaic virus (TMV)-coded movement protein (MP) with TMV RNA and short
(890 nt) synthetic RNA transcripts was visualized by atomic force microscopy on
a mica surface. MP molecules were found to be distributed along the chain of RNA
and the structure of MP-RNA complexes depended on the molar MP:RNA ratios at
which the complexes were formed. A rise in the molar MP:TMV RNA ratio from 20:1
to 60-100:1 resulted in an increase in the density of the MP packaging on TMV
RNA and structural conversion of complexes from RNase-sensitive
'beads-on-a-string' into a 'thick string' form that was partly resistant to
RNAse. The 'thick string'-type RNase-resistant complexes were also produced by
short synthetic RNA transcripts at different MP:RNA ratios. The 'thick string'
complexes are suggested to represent clusters of MP molecules cooperatively
bound to discrete regions of TMV RNA and separated by protein-free RNA segments.
Orlov V.N.,
Arutyunyan A.M., Kust S.V., Litmanovich E.A., Drachev V.A., Dobrov E.N.
Biochemistry
(Moscow) 66 (2001) 154-162
The relationship between processes of thermal
denaturation and heat-induced aggregation of tobacco mosaic virus (TMV) coat
protein (CP) was studied. Judging from differential scanning calorimetry
"melting" curves, TMV CP in the form of a trimer-pentamer mixture
("4S-protein") has very low thermal stability, with a transition
temperature at about 40 degrees C. Thermally denatured TMV CP displayed high
propensity for large (macroscopic) aggregate formation. TMV CP macroscopic
aggregation was strongly dependent on the protein concentration and solution
ionic strength. By varying phosphate buffer molarity, it was possible to merge
or to separate the denaturation and aggregation processes. Using far-UV CD
spectroscopy, it was found that on thermal denaturation TMV CP subunits are
converted into an intermediate that retains about half of its initial a-helix content and possesses high heat stability. We suppose that
this stable thermal denaturation intermediate is directly responsible for the
formation of TMV CP macroscopic aggregates.
Fedorkin
O., Solovyev A., Yelina N., Zamyatnin A. Jr, Zinovkin R., Makinen K., Schiemann
J., Morozov S.Yu.
Journal
of General Virology 82 (2001) 449-458
Complementation of movement-deficient potato
virus X (PVX) coat protein (CP) mutants, namely PVX.CP-Xho lacking the 18
C-terminal amino acid residues and PVX.DeltaCP lacking the entire CP gene, was
studied by transient co-expression with heterologous proteins. These data
demonstrated that the potyvirus CPs and both the major and minor CPs of beet
yellows closterovirus could complement cell-to-cell movement of PVX.CP-Xho but
not PVX.DeltaCP. These data also indicated that the C-terminally truncated PVX
CP lacked a movement function which could be provided in trans by the CPs of
other filamentous viruses, whereas another movement determinant specified by
some region outside the most C-terminal part of the PVX CP could not be
complemented either by potyvirus or closterovirus CPs. Surprisingly, the CP of
spherical cocksfoot mottle sobemovirus rescued all of the PVX CP movement functions,
complementing the spread of PVX.CP-Xho and, to a lesser extent, PVX.DeltaCP.
Both these mutants were also rescued by the tobacco mosaic virus (TMV) movement
protein (MP). To shed light on the movement function of PVX CP, attempts were
made to complement PVX.CP-Xho by a series of TMV MP mutants. An internal
deletion abolished complementation, suggesting that the internal region of TMV
MP, which includes a number of overlapping functional domains important for
cell-to-cell transport, provides an activity complementing movement
determinant(s) specified by the C-terminal region of PVX CP.
Agol V.I.
Molecular
Biology 35 (2001) 691-701
Picornaviruses are small animal viruses with
positive-stranded genomic RNA, which is translated using cap-independent
internal translation initiation. The key role in this is played by cis elements
of the 5'-untranslated region (5'-UTR) and, in particular, by the internal
ribosome entry site (IRES). The function of translational cis elements requires
both canonical translation initiation factors (eIFs) and additional IRES
trans-acting factors (ITAFs). All known ITAFs are cell RNA-binding proteins
which play a variety of functions in noninfected cells. Specific features of translational
cis elements substantially affect the phenotype and, in particular, tissue
tropism and pathogenic properties of picornaviruses. It is clear that, in some
cases, the molecular mechanism of this is a change in interactions between
viral cis elements and ITAFs. The properties and tissue distribution of ITAFs
may determine the biological properties of other viruses that also use the
IRES-dependent translation initiation. Since this mechanism is also involved in
translation of several cell mRNAs, ITAF may contribute to the regulation of the
most important aspects of the living activity in noninfected cells.
Lukashina
E.V., Badun G.A., Fedoseev V.M., Fedorova N.V., Ksenofontov A.L., Baratova
L.A., Dobrov E.N.
Molecular
Biology 35 (2001) 504-509
Mutant ts21-66 of the tobacco mosaic virus (TMV)
differs from the wild-type TMV-U1 by two mutations (Ile-21-->Thr and
Asp-66-->Gly) in the coat protein (CP) gene and in symptoms produced in
infected N' plants. The CP structure in TMV-U1 and ts21-66 virions was probed
by tritium planigraphy. Compared with the wild-type CP, labeling of the
N-terminal region of mutant CP was half as high and suggested its greater
shielding. A role of this CP region in virus interactions with the N'
resistance system is discussed.
Agol V.I., Belov G.A.,
Cherkasova E.A., Gavrilin G.V., Kolesnikova M.S., Romanova L.I., Tolskaya E.A.
Developments in Biologicals
105 (2001) 43-50.
Molecular
mechanisms of poliovirus reproduction in the human gut remain largely
unexplored. Nevertheless, there are grounds to believe that the virus spreads
from cell to cell, like that from person to person during natural circulation,
and involves a relatively small proportion of the highly heterogeneous viral
population generated by the previous host. This mechanism of random sampling is
responsible for the majority of fixed mutations, and contributes to the
maintenance of a certain level of viral fitness (virulence). In the long term,
random sampling may lead to the decrease in fitness and even to extinction of
some viral evolutionary branches, explaining cases of self-limiting poliovirus
infection in immunodeficient patients. A low propensity of the Sabin viruses
for natural circulation may also be a related phenomenon. The trend to decrease
in fitness may be interrupted by the appearance of rare, fitter (more virulent)
variants, which may be responsible for poliomyelitis outbreaks caused by wild
type virus, and for the development of paralytic disease in chronic carriers of
the Sabin vaccine. All these evolutionary events are largely stochastic and
hence are unpredictable in principle.
Pilipenko E.V., Viktorova
E.G., Guest S.T., Agol V.I., Roos R.P.
The EMBO Journal 20 (2001)
6899-6908
Translation
initiation of the picornavirus genome is regulated by an internal ribosome
entry site (IRES). The IRES of a neurovirulent picornavirus, the GDVII strain
of Theiler's murine encephalomyelitis virus, requires polypyrimidine
tract-binding protein (PTB) for its function. Although neural cells are
deficient in PTB, they express a neural-specific homologue of PTB (nPTB). We
now show that nPTB and PTB bind similarly to multiple sites in the GDVII IRES,
rendering it competent for efficient translation initiation. Mutation of a PTB
or nPTB site results in a more prominent decrease in nPTB than PTB binding, a
decrease in activity of nPTB compared with PTB in promoting translation
initiation, and attenuation of the neurovirulence of the virus without a marked
effect on virus growth in non-neural cells. The addition of a second-site
mutation in the mutant IRES generates a new PTB (nPTB) binding site, and
restores nPTB binding, translation initiation and neurovirulence. We conclude that
the tissue-specific expression and differential RNA-binding properties of PTB
and nPTB are important determinants of cell-specific translational control and
viral neurovirulence.
Efimov V.A., Chakhmakhcheva
O.G., Archdeacon J., Fernandez J.M., Fedorkin O.N., Dorokhov Y.L., Atabekov J.G.
Nucleic Acids Research 29
(2001) 4751-4759
An
effective procedure for specific determination of the cap structure at the 5'-terminus
of mRNA and for isolation of the corresponding full-length cDNA has been
developed. The procedure involves covalent attachment of an oligonucleotide
template extender to the 5'-cap structure of mRNA followed by RT-PCR using
M-MLV SuperScript II reverse transcriptase. In the course of reverse
transcription, the enzyme 'jumps over' the cap structure and includes the
sequence complementary to the oligonucleotide template extender into the 3'-end
of the first cDNA strand. The cap-jumping method was successfully tested using
some mammalian cellular mRNAs, genomic RNAs of tobacco mosaic virus (TMV) U1
and the recently isolated crucifer-infecting tobamovirus. Moreover, cDNA
products corresponding to the genomic tobamovirus RNA were obtained from total
RNA extracted from tobacco plants infected by crucifer-infecting tobamovirus or
tobacco mosaic virus. Using the cap-jumping method, we have shown for the first
time that genomic crucifer-infecting tobamovirus (crTMV) RNA contains a 5'-cap
structure. This improved method can be recommended for the construction of
full-length and 5'-end enriched cDNA libraries, identification of capped RNAs
and determination of their 5'-terminal sequences.
Erokhina T.N., Vitushkina
M.V., Zinovkin R.A., Lesemann D.E., Jelkmann W., Koonin E.V., Agranovsky A.A.
Journal of General Virology
82 (2001) 1983-1994
Monoclonal
antibodies (Mabs) specific to the methyltransferase (MT) and helicase (HEL)
domains of the closterovirus Beet yellows virus (BYV) were used for
immunogold labelling of ultrathin sections of virus-infected Tetragonia
expansa plants. Mabs 4A2 and 4A5 from the MT panel, and 1C4 from HEL panel,
specifically labelled distinct closterovirus-induced mambranous structures, the
‘BYV-type vesicles’, thus suggesting that the closterovirus MT-like and
HEL-like proteins co-localize in these structures. Probing of the MT and HEL
Mabs with synthetic octapeptides spanning the sequences of the recombinant MT
and HEL fragments that had been used as immunogens showed that 4A5 and 4A2
recognized a single epitope, SRLLENET (aa 686-692 in the BYV 1a protein), and
1C4 reacted with the DDPF epitope (aa 2493-2496). These epitopes apparently
reside on the exposed parts of the membrane-associated molecules of the
closterovirus MT-like and HEL-like proteins. Two other epitopes determined for
the MT Mabs that were nonreactive in the immunogold labelling, namely TMVTPGEL
(aa 750-757; Mabs 3C5, 4B4 and 4C5) and SREQLVEA (aa 806-813; Mab 2A4), are
possibly buried in the MT domain fold or shielded by membranes or the other
proteins involved in the viral replicative complex.
Ivanova O.E., Eremeeva
T.P., Karganova G.G., Rumyantsev A.A., Leshinskaya E.V., Lipskaya G.Y.,
Cherkasova E.A., Korotkova E.A., Grachev V.P., Drozdov S.G.
Developments in Biologicals
105 (2001) 219-223
After
introducing surveillance for poliomyelitis and AFP cases in the Russian
Federation in 1998, 740 AFP cases have been registered in 1998-1999, and 18 of
that number were considered as vaccine-associated paralytic poliomyelitis
(VAPP). Of 18 cases 11 were classified as VAPP of vaccine recipients and
confirmed by virus isolation; from two of the vaccine recipients virus was not
isolated, and five were poliomyelitis cases in contact non-vaccinated children.
In all the cases the disease was characterised with the typical clinical
picture with residual pareses and paralyses. One case was fatal. Vaccine virus
type 3 has been isolated from all the vaccine recipients. The MAPREC test has
shown that the quality of monovaccine type 3 bulks used for vaccinating these
children did not differ from the quality of other bulk vaccines produced by the
Chumakov Institute of Poliomyelitis. Patients surveyed for gammaglobulin were
positive. Polioviruses type 1 isolated from two of the contact cases had
changed antigenic properties and were recombinants of types 1 and 2.
Neznanov N., Kondratova A.,
Chumakov K.M., Angres B., Zhumabayeva B., Agol V.I., Gudkov A.V.
Journal of Virology 75
(2001) 10409-10420
Viral
infections often trigger host defensive reactions by activating intrinsic
(intracellular) and extrinsic (receptor-mediated) apoptotic pathways.
Poliovirus is known to encode an antiapoptotic function(s) suppressing the
intrinsic pathway. Here, the effect of poliovirus nonstructural proteins on
cell sensitivity to tumor necrosis factor (TNF)-induced (i.e.,
receptor-mediated) apoptosis was studied. This sensitivity is dramatically
enhanced by the viral proteinase 2A, due, most likely, to inhibition of
cellular translation. On the other hand, cells expressing poliovirus noncapsid
proteins 3A and 2B exhibit strong TNF resistance. Expression of 3A neutralizes
the proapoptotic activity of 2A and results in a specific suppression of TNF
signaling, including the lack of activation of NF-kB, due to elimination of the TNF
receptor from the cell surface. In agreement with this, poliovirus infection
results in a dramatic decrease in TNF receptor abundance on the surfaces of
infected cells as early as 4 h postinfection. Poliovirus proteins that confer
resistance to TNF interfere with endoplasmic reticulum-Golgi protein
trafficking, and their effect on TNF signaling can be imitated by brefeldin A,
suggesting that the mechanism of poliovirus-mediated resistance to TNF is a
result of aberrant TNF receptor trafficking.
Structure, expression and evolution of genom
Kubareva
E.A., Walter J., Vorob'eva O.V., Razumikhin M.V., Karyagina A.S., Lau P.C.,
Trautner T.
Biochemistry
(Moscow) 66 (2001) 1356-1360
A method for determination of a non-methylated
deoxycytidine (dC) residue in the recognition site of 5-cytosine DNA-methyltransferases
is suggested. The method is based on treatment of methylated DNA by sodium
bisulfite and successive reaction of the thus modified DNA with a repair
enzyme, uracil-DNA glycosylase. This method was successfully applied to
identify NlaX methyltransferase specificity.
Evstafieva
A.G., Shatsky I.N., Bogdanov A.A., Semenkov Yu.P., Vasiliev V.D.
The EMBO
Journal 1983 2(5) 799-804
Poly(U) with an average chain length of 40-70
nucleotides was modified at the 5'- or 3'-terminal residues with
2,4-dinitrophenyl derivatives. The modified poly(U) was used to form
30S.poly(U) or 70S.poly(U).Phe-tRNA complexes. Localization of the 5' and 3'
ends of the template polynucleotide on the 30S subunit and the 70S ribosome was
performed by immune electron microscopy using antibodies against dinitrophenyl
haptens. The 5' and 3' ends of poly(U) (putative entry and exit sites of the
message) were found in the same region both on the 30S subunit and the 70S
ribosome. They were located on the dorsal side of the 30S subunit between the
head and the body near the groove bordering the side ledge (platform).
Comparison of the size of this region with the possible length of the
polynucleotide chain covered by the ribosome allowed us to suggest that the
message makes a 'U-turn" (or forms a 'loop') as it passes through the
ribosome.
Bakeeva
L.E., Zamyatnina V.A., Shorning B.Y., Aleksandrushkina N.I., Vanyushin B.F.
Biochemistry
(Moscow) 66 (2001) 850-859
Ionol (BHT), a compound having antioxidant
activity, at concentrations in the range 1-50 mg/liter (0.45x10-5 -
2.27x10-4 M), inhibits growth of etiolated wheat seedlings, changes
the morphology of their organs, prolongs the coleoptile life span, and prevents
the appearance of specific features of aging and apoptosis in plants. In
particular, BHT prevents the age-dependent decrease in total DNA content,
apoptotic internucleosomal fragmentation of nuclear DNA, appearance in the cell
vacuole of specific vesicles with active mitochondria intensively producing
mtDNA, and formation of heavy mitochondrial DNA rho = 1.718 g/cm3)
in coleoptiles of etiolated wheat seedlings. BHT induces large structural
changes in the organization of all cellular organelles (nucleus, mitochondria,
plastids, Golgi apparatus, endocytoplasmic reticulum) and the formation of new
unusual membrane structures in the cytoplasm. BHT distorts the division of
nuclei and cells, and this results in the appearance of multi-bladed polyploid
nuclei and multinuclear cells. In roots of etiolated wheat seedlings, BHT
induces intensive synthesis of pigments, presumably carotenoids, and the
differentiation of plastids with formation of chloro- or chromoplasts. The
observed multiple effects of BHT are due to its antioxidative properties (the
structural BHT analog 3,5-di-tert-butyltoluene is physiologically inert; it has
no effect similar to that of BHT). Therefore, the reactive oxygen species (ROS)
controlled by BHT seem to trigger apoptosis and the structural reorganization
of the cytoplasm in the apoptotic cell with formation of specific vacuolar
vesicles that contain active mitochondria intensively producing mtDNA. Thus,
the inactivation of ROS by BHT may be responsible for the observed changes in the
structure of all the mentioned cellular organelles. This corresponds to the
idea that ROS control apoptosis and mitosis including formation of cell wall,
and they are powerful secondary messengers that regulate differentiation of
plastids and the Golgi apparatus in plants.
Shorning
B.Yu., Vanyushin B.F.
Biochemistry
(Moscow) 66 (2001) 753-762
By computer analysis of the known data bases, we
have established that the open reading frames (ORF) coding for proteins that
possess high degree of homology with procaryotic DNA-(amino)methyltransferases
are present in the genomes of Leishmania major, Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Arabidopsis thaliana, Drosophila melanogaster,
Caenorhabditis elegans, and Homo sapiens. Conservative motifs typical for
bacterial DNA-(amino)methyltransferases are detected in the amino acid
sequences of these putative proteins. The ORF of all putative eucaryotic
DNA-(amino)methyltransferases found are encoded in nuclear DNA. In
mitochondrial genomes including a few fully sequenced higher plant mtDNA,
nucleotide sequences significantly homologous to genes of procaryotic
DNA-(amino)methyltransferases are not found. Thus, ORF homologous to bacterial
adenine DNA-methyltransferases are present in nuclei of protozoa, yeasts,
insects, nematodes, vertebrates, higher plants, and other eucaryotes. A special
search for corresponding proteins and, in particular, adenine
DNA-methyltransferases in these organisms and a study of their functions are
quite promising.
Milyutina
I.A., Aleshin V.V., Mikrjukov K.A., Kedrova O.S., Petrov N.B.
Gene 272
(2001) 131-139
In order to ascertain a phylogenetic position of
the freshwater amitochondriate amoeboflagellate Pelomyxa palustris its
small subunit (SSU) rRNA gene was amplified and sequenced. It was shown to be
3502 bp long. The predicted secondary structure of its rRNA includes at least
16 separate expansion zones located in all the variable regions (V1-V9), as
well as in some conservative gene regions. Most insertions are represented by
sequences of low complexity that have presumably arisen by a slippage
mechanism. Relatively conservative, uniformly positioned motifs contained in
regions V4 and V7, as well as in some others, made it possible to perform
folding. In maximum likelihood, maximum parsimony, and neighbor-joining trees, P.
palustris tends to cluster with amitochondriate and secondary lost
mitochondria amoebae and amoeboflagellates Entamoeba, Endolimax nana,
and Phreatamoeba balamuthi, comprising together with them and aerobic
lobose amoebae Vannella, Acanthamoeba, Balamuthia, and Hartmannella
a monophyletic cluster. Another pelobiont, Mastigamoeba invertens, does
not belong to this cluster. No specific similarity was discovered between the
SSU rRNA of P. palustris and amitochondriate taxa of 'Archezoa': Diplomonada,
Parabasalia, Microsporidia. Pelomyxa palustris SSU rRNA
does not occupy a basal position in the phylogenetic trees and could be
ascribed to the so-called eukaryotic 'crown' group if the composition of the
latter were not so sensitive to the methods of tree building. Thus, molecular
and morphological data suggest that P. palustris represents a
secondarily modified eukaryotic lineage.
Andreev
S.Yu., Antsypovich S.I., Volkov E.M., Romanova E.A., Hianik T., Oretskaia T.S.
Russian
Journal of Bioorganic Chemistry 27 (2001) 210-216
A method for directional introduction of
oleylamine residues to any position of oligodeoxyribonucleotides during their
automated synthesis was developed. The presence of oleylamine residues in 3'-
or 5'-terminal nucleotides was shown to have no effect on the thermodynamic
stability of DNA duplexes formed by such oligonucleotides and the complementary
sequences. The rate of the snake venom phosphodiesterase hydrolysis of
oligonucleotides containing oleylamine residues in the 3'-terminal units was
shown to be markedly lower than that of natural oligonucleotides.
Rodina
E.V., Vainonen Y.P., Vorobyeva N.N., Kurilova S.A., Nazarova T.I., Avaeva S.M.
European
Journal of Biochemistry / FEBS 268 (2001) 3851-3857
Excess of Mg2+ ions is known to
inhibit the soluble inorganic pyrophosphatases (PPases). In contrast, the
mutant Escherichia coli inorganic pyrophosphatase Asp42-->Asn is
three times more active than native and retains its activity at high Mg2+
concentration. In this paper, another two mutant variants with Asp42 replaced
by Ala or Glu were investigated to characterize the role of Asp42 in catalysis.
pH-independent kinetic parameters of MgPPi hydrolysis and the
dissociation constants for the activating and inhibitory Mg2+ ions
were calculated. It was shown that Mg2+ inhibition of MgPPi
hydrolysis by native PPase exhibited uncompetitive kinetics under the
saturating substrate concentration. All three substitutions of Asp42 lead to a
sharp decrease of inhibitory Mg2+ affinity to the enzyme. These
findings allow determination of the sites of inhibitory and substrate Mg2+
ions binding to PPase. Common features of these mutants allow the
conclusion that the function of Asp42 is to accurately coordinate the residues
implicated in the substrate and the inhibitory Mg2+ ion binding to
PPase active site. Structural analysis of PPase complexed with Mg2+
compared with PPase complexed with Mn2+ and reaction products
confirms this supposition.
Gulevich
A.Yu., Yusupova R.A., Drygin Yu.F.
Biochemistry
(Moscow) 66 (2001) 345-349
The
substrate specificity of the "unlinking" enzyme from ascite carcinoma
Krebs II cells has been investigated. The enzyme specifically splits the
interpolymeric phosphodiester bond between Kp and the 5;-terminal phosphate
group of the uridylic acid residue in the Kp-pUpUpGp complex.
Shatskii
I.N.
Molecular
Biology 35 (2001) 628-637.
Papers on the mechanisms of translation
initiation in mammals studied by reconstruction of initiation complexes from
individual components are reviewed. The author points to the constraints of this
approach and to the pitfalls ignoring which one might come to erroneous
conclusions and even artifacts. In addition, some methods employed in the field
as well as some technical problems are discussed in the paper, together with
the means of obviating them. The review could be a guidebook for newcomers into
this quite labor-consuming field.
Zatsepin
T.S., Kachalova A.V., Romanova E.A., Stestenko D.A., Gait M.J., Oretskaia T.S.
Russian
Journal of Bioorganic Chemistry 27 (2001) 45-51
A new uridine derivative,
2'-O-(2,3-dihydroxypropyl)uridine, and its 3'-phosphoramidite were obtained for
direct introduction into oligonucleotides during automated chemical synthesis.
Oligonucleotides 10 to 15 nt long harboring 2'-O-(2,3-dihydroxypropyl)uridine
residues were synthesized; periodate oxidation of these oligomers gave
oligonucleotides containing 2'-O-(2-oxoethyl)uridine residues. The presence of
a reactive aldehyde group in 2' position of the carbohydrate moiety was
confirmed by the interaction with p-nitrophenylhydrazine and methionine methyl
ester. The thermostability of DNA duplexes containing modified units is practically
indistinguishable from that of the natural analogues.
Osipova E.S., Kokaeva Z.G.,
Troitskii A.V., Dolgikh Y.I., Shamina Z.B., Gostimskii S.A.
Genetika 37 (2001) 91-96
The
genetic difference between maize line A188 and A188-derived somaclones was
assessed via analysis of randomly amplified polymorphic DNA (RAPD). In total,
15 out of 17 decanucleotide primers used each allowed amplification of 2-17
fragments ranging 200-2000 bp. The RAPD patterns did not differ between
individual plants of line A188, which demonstrated again its high genetic
homogeneity. The difference between the initial line and the somaclones was
high, ranging 64-74%. On evidence of the genetic divergence, the somaclones
formed two clusters. The distribution of somaclones between these clusters was
consistent with their origin.
Brodin P.,
Pinskaya M., Parsch U., Bischerour J., Leh H., Romanova E., Engels J.W.,
Gottikh M., Mouscadet J.F.
Nucleosides Nucleotides
Nucleic Acids 20 (2001) 481-486
Integration
of the proviral DNA into the genome of infected cells is a key step of HIV-1
replication. Integration is catalyzed by the viral enzyme integrase (IN).
6-oxocytidine-containing oligonucleotides were found to be efficient inhibitors
of integrase in vitro. The inhibitory effect is sequence-specific and strictly
requires the presence of the 6-oxocytidine base. It is due to the impairment of
the integrase binding to its substrate and does not involve an auto-structure
of the oligonucleotide.
Hianik T., Gajdos V., Krivanek
R., Oretskaya T., Metelev V., Volkov E., Vadgama P.
Bioelectrochemistry 53
(2001) 199-204
We tested the
possibility of amperometric detection of DNA hybridization on a gold surface
influenced by the immobilization of oligonucleotide giving different orientations
of single stranded DNA relative to the gold surface. The DNA sensor was
fabricated by chemisorption of 18-mer oligonucleotide modified by a
phosphorothioate group either at its 3' or both 3' and 5' terminal. After
immobilization of oligonucleotide to the gold support, the sensor was immersed
in 11-mercaptoundecanoic acid (MUA) solution. Further chemisorption of MUA
resulted in approximately 10-fold increase of resistance of the organic layer.
Addition of complementary oligonucleotide resulted in an increase of
conductivity for DNA sensor oriented perpendicular to the gold support (DNA
with one thiol group), while the conductance decreased for DNA sensor with
single stranded DNA oriented parallel to the gold support (with DNA modified by
thiol groups at both 3' and 5' terminals). Addition of non-complementary chain
resulted a slight decrease or no change of sensor conductivity. The
hybridization process at both types of DNA orientations is not cooperative and
can be described by Langmuir isotherms. The hybridization event on gold support
has been confirmed by mass detection using the quartz crystal microbalance
technique.
Determination
of biologically active low-molecular-mass thiolsinhuman blood. III. Highly
sensitive narrow-bore isocraticreversedphase high-performance liquid
chromatographywithfluorescence detection.
Ivanov
A.R., Nazimov I.V., Baratova L., Lobazov A.P., Popkovich G.B.
Journal of Chromatography.
A. 913 (2001) 315-318
The
fast isocratic narrow-bore reversed-phase high-performance liquid
chromatographic method employing fluorescence detection is described for the
precise reproducible simultaneous measurement of total homocysteine, cysteine
and glutathione in human blood. Sample preparation involves conversion of
disulfides to free thiols with triphenylphosphine, precipitation of proteins
with sulfosalicylic acid, and conjugation of thiols with monobromobimane.
Optimized sample preparation conditions as well as chromatographic conditions
allowed to obtain reliable quantitative results within the concentration range
corresponding to the levels of these thiols in human blood in norm and
pathology. The detection limit was approximately 70 amol for all labeled
aminothiols. The proposed method for these compounds analysis includes simple sample
preparation, high selectivity, good linearity (r2>0.998), high
reproducibility (within-run precision for derivatized aminothiol peaks area
RSD<1.8% for three times consequently injected sample); high reliability and
the small sample volume (1 ml) required for analysis make it suitable for clinical
studies.
Ostareck D.H.,
Ostareck-Lederer A., Shatsky I.N., Hentze M.W.
Cell 104 (2001) 281-290
15-lipoxygenase
(LOX) expression is translationally silenced in early erythroid precursor cells
by a specific mRNA-protein complex formed between the differentiation control
element in the 3' untranslated region (UTR) and hnRNPs K and E1. The 3'UTR
regulatory complex prevents translation initiation by an unknown mechanism. We
demonstrate that the 40S ribosomal subunit can be recruited and scan to the
translation initiation codon even when the silencing complex is bound to the
3'UTR. However, the joining of the 60S ribosomal subunit at the AUG codon to
form a translation competent 80S ribosome is inhibited, unless initiation is
mediated by the IGR-IRES of the cricket paralysis virus. These findings
identify the critical step at which LOX mRNA translation is controlled and
reveal that 60S subunit joining can be specifically regulated.
Pestova T.V., Kolupaeva
V.G., Lomakin I.B., Pilipenko E.V., Shatsky I.N., Agol V.I., Hellen C.U.
Proc Natl Acad Sci U S A 98
(2001) 7029-7036
Translation
initiation is a complex process in which initiator tRNA, 40S, and 60S ribosomal
subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S
ribosome at the initiation codon of mRNA. The cap-binding complex eIF4F and the
factors eIF4A and eIF4B are required for binding of 43S complexes (comprising a
40S subunit, eIF2/GTP/Met-tRNAi and eIF3) to the 5' end of capped mRNA but are
not sufficient to promote ribosomal scanning to the initiation codon. eIF1A
enhances the ability of eIF1 to dissociate aberrantly assembled complexes from
mRNA, and these factors synergistically mediate 48S complex assembly at the
initiation codon. Joining of 48S complexes to 60S subunits to form 80S
ribosomes requires eIF5B, which has an essential ribosome-dependent GTPase
activity and hydrolysis of eIF2-bound GTP induced by eIF5. Initiation on a few
mRNAs is cap-independent and occurs instead by internal ribosomal entry.
Encephalomyocarditis virus (EMCV) and hepatitis C virus epitomize distinct
mechanisms of internal ribosomal entry site (IRES)-mediated initiation. The
eIF4A and eIF4G subunits of eIF4F bind immediately upstream of the EMCV
initiation codon and promote binding of 43S complexes. EMCV initiation does not
involve scanning and does not require eIF1, eIF1A, and the eIF4E subunit of
eIF4F. Initiation on some EMCV-like IRESs requires additional noncanonical
initiation factors, which alter IRES conformation and promote binding of
eIF4A/4G. Initiation on the hepatitis C virus IRES is even simpler: 43S
complexes containing only eIF2 and eIF3 bind directly to the initiation codon
as a result of specific interaction of the IRES and the 40S subunit.
Metelev V.G., Borisova
O.A., Volkov E.M., Oretskaya T.S., Dolinnaya N.G.
Nucleic Acids Research 29
(2001) 4062-4069
Novel modified DNA
duplexes with single bridging 5'-SS-monophosphoryldithio links
[-OP(=O)-O(-)-SS-CH(2)-] were synthesized by autoligation of an oligonucleotide
3'-phosphorothioate and a 5'-mercapto-oligonucleotide previously converted to a
2-pyridyldisulfide adduct. Monophosphoryldisulfide link formation is not a stringent
template-dependent process under the conditions used and does not require
strong binding of the reactive oligomers to the complementary strand. The
modified internucleotide linkage, resembling the natural phosphodiester bond in
size and charge density, is stable in water, easily undergoes thiol-disulfide
exchange and can be specifically cleaved by the action of reducing reagents.
DNA molecules containing an internal -OP(=O)-O(-)-SS-CH(2)- bridge are stable
to spontaneous exchange of disulfide-linked fragments (recombination) even in
the single-stranded state and are promising reagents for autocrosslinking with
cysteine-containing proteins. The chemical and supramolecular properties of
oligonucleotides with 5'-sulfhydryl groups were further characterized. We have
shown that under the conditions of chemical ligation the 5'-SH group of the
oligonucleotide has a higher reactivity towards
N-hydroxybenzotriazole-activated phosphate in an adjacent oligonucleotide than
does the OH group. This autoligation, unlike disulfide bond formation, proceeds
only in the presence of template oligonucleotide, necessary to provide the
activated phosphate in close proximity to the SH-, OH- or phosphate function.
Kuznetsov G.V., Kulikov
E.E., Petrov N.B., Ivanova N.V., Lomov A.A., Kholodova M.V., Poltaraus A.B.
Naturwissenschaften 88
(2001) 123-125.
The
controversial phylogenetic position of the recently described South-East Asian
endemic bovid, Pseudonovibos spiralis, was evaluated on the basis of
phylogenetic analyses of originally obtained nearly complete 12S mitochondrial
rDNA sequences for this species and Bubalus bubalis and 26 sequences of Bovidae
from the Genbank using Cervus elaphus (Cervidae) as outgroup. In
most of the phylogenetic analyses performed using PAUP 4.0 (maximum parsimony,
maximum likelihood and neighbour-joining), Bovidae consisted of two
major clades: Bovinae including the tribes Bovini, Tragelaphini
and Boselaphini, and Antilopinae + Caprinae, incorporating
all other bovids. In most trees P. spiralis fell within the buffalos
(subtribe Bovina) between Bubalus and Syncerus. Therefore,
our phylogenetic analyses of bovid mitochondrial 12S rRNA gene sequences
suggest the close relationship of this enigmatic species with the buffalos and
its placement within the subtribe Bovina.
Petrov
A.V., Dmitriev P.V., Sokolov K.A., Dontsova O.A.
Biochemistry
(Moscow) (2001) 66 1361-1367
The telomere DNA of most eucaryotes consists of
tandem DNA repeats and a number of associated proteins. The synthesis of the
G_rich DNA strand is performed by the telomerase complex. The complementary
C_strand is synthesized by DNA_dependent DNA polymerases. Using telomerase
reverse transcriptase tagging followed by immunoprecipitation coupled with
two_step purification, we have found a specific DNA_dependent DNA polymerase
activity associated with telomerase of Saccharomyces cerevisiae. Investigation
of the biochemical properties of this DNA polymerase activity confirms the
hypothesis of tight interaction of DNA polymerase with telomerase.
Zvereva
M.I., Ivanov P.V., Teraoka Y., Topilina N.I., Dontsova O.A., Bogdanov A.A.,
Kalkum M., Nierhaus K.H., Shpanchenko O.V.
The
Journal of Biological Chemistry 276 (2001) 47702-47708
Transfer-messenger RNA (tmRNA) is a stable RNA
in bacteria of 360 ± 40 nucleotides that can be charged with alanine
and can function as both tRNA and mRNA. Ribosomes that are stalled either in a
coding region of mRNA or at the 3' end of an mRNA fragment lacking a stop codon
are rescued by replacing their mRNA for tmRNA. Here we demonstrate that the
interaction of tmRNA with the elongation factor Tu shows unexpected features.
Deacylated tmRNA can form a complex with either EF-Tu.GDP or EF-Tu.GTP, the
association constants are about one order of magnitude smaller than that of an
Ala-tRNA.EF-Tu.GTP complex. tmRNA as well as Ala-tmRNA can be efficiently
cross-linked with EF-Tu.GDP using a zero-length cross-link. The efficiency of
cross-linking in the case of deacylated tmRNA does not depend on an intact
CCA-3' end and is about the same, regardless whether protein mixtures such as
the post-ribosomal supernatant (S100 enzymes) or purified EF-Tu are present.
Two cross-linking sites with EF-Tu.GDP have been identified that are located
outside the tRNA part of tmRNA, indicating an unusual interaction of tmRNA with
EF-Tu.GDP.
Matadeen
R., Sergiev P., Leonov A., Pape T., van der Sluis E., Mueller F., Osswald M.,
von Knoblauch K., Brimacombe R., Bogdanov A., van Heel M., Dontsova O.
Journal
of Molecular Biology 307 (2001) 1341-1349
Insertions were introduced by a two-step
mutagenesis procedure into each of five double-helical regions of Escherichia
coli 23 S rRNA, so as to extend the helix concerned by 17 bp. The helices
chosen were at sites within the 23 S molecule (h9, h25, h45, h63 and h98) where
significant length variations between different species are known to occur. At
each of these positions, with the exception of h45, there are also significant
differences between the 23 S rRNAs of E. coli and Haloarcula
marismortui. Plasmids carrying the insertions were introduced into an E.
coli strain lacking all seven rrn operons. In four of the five cases the
cells were viable and 50 S subunits could be isolated; only the insertion in
h63 was lethal. The modified subunits were examined by cryo-electron microscopy
(cryo-EM), with a view to locating extra electron density corresponding to the
insertion elements. The results were compared both with the recently determined
atomic structure of H. marismortui 23 S rRNA in the 50 S subunit, and
with previous 23 S rRNA modelling studies based on cryo-EM reconstructions of E.
coli ribosomes. The insertion element in h45 was located by cryo-EM at a
position corresponding precisely to that of the equivalent helix in H.
marismortui. The insertion in h98 (which is entirely absent in H.
marismortui) was similarly located at a position corresponding precisely to
that predicted from the E. coli modelling studies. In the region of h9,
the difference between the E. coli and H. marismortui secondary
structures is ambiguous, and the extra electron density corresponding to the
insertion was seen at a location intermediate between the position of the
nearest helix in the atomic structure and that in the modelled structure. In
the case of h25 (which is about 50 nucleotides longer in H. marismortui),
no clear extra cryo-EM density corresponding to the insertion could be
observed.
Ledneva
R.K., Alekseevskii A.V., Vasil'ev S.A., Spirin S.A., Kariagina A.S.
Molecular
Biology 35 (2001) 764-777
This review is devoted to the structural aspects
of interaction of homeodomains with DNA. Presented are the list of all
homeodomains with known spatial structure and the alignment of their amino acid
sequences. The structure of homeodomains and contacts of their amino acid
residues with DNA bases and sugar-phosphate backbone are described. The role of
water molecules in DNA binding is discussed. Structures of multicomponent
protein complexes on DNA including homeodomains are characterized.
Enzymology and
biotechnology
Functional flexibility of the
transketolase molecule.
Kochetov
G.A.
Biochemistry
(Moscow) 66 (2001) 1077-1085
Transketolase is the simplest representative of
the thiamine diphosphate-dependent enzymes. It was the first of these enzymes
for which X-ray analysis was performed. Based on the data of X-ray studies and
using the mutagenesis technique, the nature of functional groups of the enzyme
involved in the interaction with substrates and cofactors and in the coenzyme activation
was defined. Thus, considerable achievements have been made in studying the
structure of transketolase. However, there is relatively little information on
the conformational flexibility of the enzyme molecule while it is functioning,
i.e., during its interaction with cofactors and substrates and in the course of
intermediate product formation. This review summarizes mainly the results
obtained in the author's group, as well as those rare data on this subject that
could be found in literature.
Nagradova
N.K.
Biochemistry
(Moscow) 66 (2001) 1067-1076
The properties of the active center of
phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are
considered with emphasis on the structure of anion-binding sites and their role
in catalysis. The results of studies on the molecular mechanism of the effect
of NAD+ on the enzyme conformation are discussed. Experimental
evidence is presented supporting the idea that negative cooperativity of NAD+
binding and half-of-the-sites reactivity exhibited by GAPDH are generated by
different mechanisms. Data obtained with rabbit muscle and Escherichia coli
GAPDH point to preexisting asymmetry in these tetramers. Structural
determinants that can control the transition of the tetramer from the symmetric
to the asymmetric state were found.
Danshina
P.V., Schmalhausen E.V., Avetisyan A.V., Muronetz V.I.
IUBMB
Life 51 (2001) 309-314
Influence of H2O2 on
glycolysis was investigated. A hypothesis previously formulated was tested
according to which a mild oxidation of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) results in uncoupling of oxidation and phosphorylation at this step of
glycolysis due to acylphosphatase activity of the oxidized enzyme. Incubation
of a mixture of purified glycolytic enzymes, as well as a muscle extract, in
the presence of 10-100 mM H2O2 was shown to result
in an increase in the rate of glycolysis. The level of lactate accumulation in
the oxidized samples increased by 80-150% compared to the samples containing
mercaptoethanol. No ATP was formed by the H2O2-stimulated
glycolysis. Thus, H2O2 really caused uncoupling of
oxidation and phosphorylation in glycolysis. A role of GAPDH oxidation in
regulation of glycolysis is discussed.
Muronetz
V.I., Sholukh M., Korpela T.
Journal
of Biochemical and Biophysical Methods 49 (2001) 29-47
Biospecific recognition between proteins is a
phenomenon that can be exploited for designing affinity-chromatographic
purification systems for proteins. In principle, the approach is straightforward,
and there are usually many alternative ways, since a protein can be always
found which binds specifically enough to the desired protein. Routine
immunoaffinity chromatography utilizes the recognition of antigenic epitopes by
antibodies. However, forces involved in protein-protein interactions as well
the forces keeping the three-dimensional structures of proteins intact are
complicated, and proteins are easily unfolded by various factors with
unpredictable results. Because of this and because of the generally high
association strength between proteins, the correct adjustment of binding forces
between an immobilized protein and the protein to be purified as well as the
release of bound proteins in biologically active form from affinity complexes
are the main problem. Affinity systems involving interactions like
enzyme-enzyme, subunit-oligomer, protein-antibody, protein-chaperone and the
specific features involved in each case are presented as examples. This article
also aims to sketch prospects for further development of the use of
protein-protein interactions for the purification of proteins.
Yazykova
M.Yu., Muronets V.I.
Applied
Biochemistry and Microbiology 37 (2001) 174-177
Lactate dehydrogenase (EC 1.1.1.27) and
dithiothreitol (DTT) were coimmobilized on Sepharose activated with cyanogen
bromide. It was demonstrated that the addition of 10 mM DTT (but not
2-mercaptoethanol) during immobilization increased the enzyme specific activity
1.5–5-fold depending on the initial extent of Sepharose activation by cyanogen
bromide. The total activity increased two- to threefold. The lactate
dehydrogenase preparations were rich in matrix-immobilized sulfhydryl groups
(1.8–13.0 nmol per ml gel). The presence of DTT increased the stability of
immobilized lactate dehydrogenase.
Sud'ina
G.F., Brock T.G., Pushkareva M.A., Galkina S.I., Turutin D.V., Peters-Golden
M., Ullrich V.
The
Biochemical Journal 359 (2001) 621-629
Sulphatides are sulphate esters of
galactocerebrosides that are present on the surfaces of many cell types and act
as specific ligands to selectins. The present study was undertaken to
investigate the effect of sulphatides on polymorphonuclear granulocyte (PMN)
attachment, spreading and 5-lipoxygenase (5-LO) metabolism. Sulphatides, but
not non-sulphated galactocerebrosides, dose-dependently enhanced attachment to
collagen, as measured by the myeloperoxidase assay. Studies with blocking
antibodies indicated that the increased attachment was mediated by CD11b/CD18
(Mac-1) b 2 integrin. Scanning electron microscopy indicated
that sulphatides also greatly enhanced the degree of cell spreading. In PMNs
treated in suspension, sulphatides had no effect on the ionophore
A23187-stimulated release of arachidonic acid and the synthesis of 5-LO
metabolites. In contrast, in PMNs attached to collagen, the enzymic conversion
of arachidonic acid by 5-LO was inhibited by sulphatides. Inhibition of 5-LO
metabolism by sulphatides was observed even in the presence of exogenous
substrate, suggesting that sulphatides directly inhibited 5-LO action.
Consistent with this, sulphatides interfered with ionophore-induced
translocation of the 5-LO to the nuclear envelope. Substances competing with
sulphatide binding to cells, like dextran sulphate, or a strong inhibitor of
cell spreading, like the actin-polymerizing agent jasplakinolide, prevented the
effects of sulphatides on PMN attachment and spreading and leukotriene
synthesis. We conclude that shape changes occurring in response to sulphatides
specifically impair PMN leukotriene synthesis by inhibiting translocation of
5-LO.
Solov'eva
O.N., Bykova I.A., Meshalkina L.E., Kovina M.V., Kochetov G.A.
Biochemistry
(Moscow) 66 (2001) 932-936
The interaction of transketolase ketosubstrates
with the holoenzyme has been studied. On addition of ketosubstrates cleaving
both irreversibly (hydroxypyruvate) and reversibly (xylulose 5-phosphate),
identical changes in the CD spectrum at 300-360 nm are observed. The changes in
this spectral region, as previously shown, are due to the formation of the
catalytically active holoenzyme from the apoenzyme and the coenzyme, and the
cleavage of ketosubstrates by transketolase. The identity of the changes in
transketolase CD spectrum caused by the addition of reversibly or irreversibly
cleaving substrates indicates that in the both cases the changes are due to the
formation of an intermediate product of the transketolase reaction--a
glycolaldehyde residue covalently bound to the coenzyme within the holoenzyme
molecule. Usually, in the course of the transferase reaction, the
glycolaldehyde residue is transferred to an aldose (acceptor substrate),
resulting in the recycling of the holoenzyme free of the glycolaldehyde
residue. The removal of the glycolaldehyde residue from the holoenzyme appears
to proceed even in the absence of an aldose. However, the glycolaldehyde cannot
be found the free state because it condenses with another glycolaldehyde
residue formed in the course of the cleavage of another ketosubstrate molecule
yielding erythrulose.
Galkina
S.I., Sud'ina G.F., Ullrich V.
Experimental
Cell Research 266 (2001) 222-228
Human neutrophils developed long thin
tubulovesicular extensions (cytonemes) upon adhesion to fibronectin-coated
substrata, when spreading was blocked. We observed extension formation when
neutrophils were plated to fibronectin-coated substrata in Na+-free
extracellular medium or in the presence of drugs capable of inhibiting
spreading: 4-bromophenacyl bromide, N-ethylmaleimide,
7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and cytochalasin D. Addition of Na+
ions or washing of inhibitors restored neutrophil spreading. Phase-contrast and
scanning electron microscopy revealed two types of extensions: (1) highly
dynamic, flexible tubulovesicular extensions with unattached tips 0.2-0.4 mm in diameter, which can achieve 70-80 mm in length during 20 min, and (2) thinner straight extensions
with flattened tips, which were formed in the presence of phorbol 12-myristate
13-acetate and connected cells to substratum or to the neighboring cells
several cell diameters away. The latter may have derived from the former
through tension after attachment of the tips. Spreading and extension formation
may represent two states of the cell adhesive and communicative mechanism.
Parfenyev
A.N., Salminen A., Halonen P., Hachimori A., Baykov A.A., Lahti R.
Journal
of Molecular Biology 276 (2001) 24511-24518
Pyrophosphatase (PPase) from Bacillus
subtilis has recently been found to be the first example of a family II
soluble PPase with a unique requirement for Mn2+. In the present
work, we cloned and overexpressed in Escherichia coli putative genes for
two more family II PPases (from Streptococcus mutans and Streptococcus
gordonii), isolated the recombinant proteins, and showed them to be highly
specific and active PPases (catalytic constants of 1700-3300 s-1 at
25 degrees C in comparison with 200-400 s-1 for family I). All three
family II PPases were found to be dimeric manganese metalloenzymes,
dissociating into much less active monomers upon removal of Mn2+.
The dimers were found to have one high affinity manganese-specific site Kd
of 0.2-3 nm for Mn2+ and 10-80 mm for Mg2+) and two or three moderate
affinity sites Kd approximately 1 mm for both cations) per subunit.
Mn2+ binding to the high affinity site, which occurs with a
half-time of less than 10 s at 1.5 mm Mn2+, dramatically shifts the
monomer <--> dimer equilibrium in the direction of the dimer, further
activates the dimer, and allows substantial activity (60-180 s-1)
against calcium pyrophosphate, a potent inhibitor of family I PPases.
Strokin
M.L., Sergeeva M.G., Mevkh A.T., Varfolomeyev S.D.
Biochemistry
(Moscow) 66 (2001) 312-318
The kinetics of 3H-labeled arachidonic acid (AA,
10-10-10-5 M) incorporation into murine peritoneal
macrophages was investigated. During the incorporation of AA into the cells,
the steady state was reached at 10 h. The level of incorporation consisted of
48-50% for nanomolar concentrations and 28-30% for mmolar concentrations of AA. Exogenous AA in mmolar but not nanomolar concentrations stimulated [3H]AA
release from intracellular stores of pre-labeled cells. A mathematical model
fitting the behavior of the experimental system is proposed. The difference in
the level of uptake of AA in nanomolar and mmolar concentrations is explained by the
activation of AA release from intracellular stores at high concentrations of
exogenous AA.
Youshko
M.I., van Langen L.M., de Vroom E., van Rantwijk F., Sheldon R.A., SvedasV.K.
Biotechnology
and Bioengineering 73 (2001) 426-430
The synthesis of ampicillin catalyzed by Escherichia
coli penicillin acylase was optimized in an aqueous system with partially
dissolved antibiotic nucleus 6-aminopenicillanic acid (6-APA). The yields of
both 6-APA and acyl donor could be improved by repetitively adding substrates
to the reaction, allowing the concentration of 6-APA to remain saturated
throughout. In this reaction concept, with four subsequent additions of
substrates, 97% conversion of 6-APA and 72% of D-(-)-phenylglycine methyl ester
(D-PGM) to ampicillin was achieved. The synthetic potential of this concept was
estimated using a mathematical model which showed that by increasing the amount
of added substrates a nearly quantitative conversion of 6-APA and 85%
conversion of acyl donor into ampicillin could be achieved.
Zyryanov
A.B., Pohjanjoki P., Kasho V.N., Shestakov A.S., Goldman A., Lahti R., Baykov
A.A.
The
Journal of Biological Ñhemistry 276 (2001) 17629-17634
Binding
of pyrophosphate or two phosphate molecules to the pyrophosphatase (PPase)
active site occurs at two subsites, P1 and P2. Mutations at P2 subsite residues
(Y93F and K56R) caused a much greater decrease in phosphate binding affinity of
yeast PPase in the presence of Mn2+ or Co2+ than mutations at P1 subsite
residues (R78K and K193R). Phosphate binding was estimated in these experiments
from the inhibition of ATP hydrolysis at a sub-Km concentration of ATP. Tight
phosphate binding required four Mn2+ ions/active site. These data identify P2
as the high affinity subsite and P1 as the low affinity subsite, the difference
in the affinities being at least 250-fold. The time course of five
"isotopomers" of phosphate that have from zero to four 18O during
[18O]Pi-[16O]H2O oxygen exchange indicated that the phosphate containing added
water is released after the leaving group phosphate during pyrophosphate
hydrolysis. These findings provide support for the structure-based mechanism in
which pyrophosphate hydrolysis involves water attack on the phosphorus atom
located at the P2 subsite of PPase.
Bykova
I.A., Solovjeva O.N., Meshalkina L.E., Kovina M.V., Kochetov G.A.
Biochemical
and biophysical research communications 280 (2001) 845-847
Apart from catalyzing the common two-substrate
reaction with ketose as donor substrate and aldose as acceptor substrate,
transketolase is also able to catalyze a one-substrate reaction utilizing only
ketose (xylulose 5-phosphate) as substrate. The products of this one-substrate
reaction were glyceraldehyde 3-phosphate and erythrulose. No free
glycolaldehyde (a product of xylulose 5-phosphate splitting in the
transketolase reaction) was revealed.
Pohjanjoki
P., Fabrichniy I.P., Kasho V.N., Cooperman B.S., Goldman A., Baykov A.A., Lahti
R.
The
Journal of Biological Ñhemistry 276 (2001) 434-441
The pattern of yeast pyrophosphatase (Y-PPase)
inhibition by fluoride suggests that it replaces active site Mg2+-bound
nucleophilic water, for which two different locations were proposed previously.
To localize the bound fluoride, we investigate here the effects of mutating
Tyr(93) and five dicarboxylic amino acid residues forming two metal binding
sites in Y-PPase on its inhibition by fluoride and its five catalytic functions
(steady-state PPi hydrolysis and synthesis, formation of
enzyme-bound PPi at equilibrium, phosphate-water oxygen exchange,
and Mg2+ binding). D117E substitution had the largest effect on
fluoride binding and made the P-O bond cleavage step rate-limiting in the
catalytic cycle, consistent with the mechanism in which the nucleophile is
coordinated by two metal ions and Asp(117). The effects of the mutations on PPi
hydrolysis (as characterized by the catalytic constant and the net rate
constant for P-O bond cleavage) were in general larger than on PPi
synthesis (as characterized by the net rate constant for PPi release
from active site). The effects of fluoride on the Y-PPase variants confirmed
that PPase catalysis involves two enzyme.PPi intermediates, which
bind fluoride with greatly different rates (Baykov, A. A., Fabrichniy, I. P.,
Pohjanjoki, P., Zyryanov, A. B., and Lahti, R. (2000) Biochemistry 39,
11939-11947). A mechanism for the structural changes underlying the
interconversion of the enzyme.PPi intermediates is proposed.
Toward
a quantum-mechanical description of metal-assisted phosphoryl transfer in
pyrophosphatase
Heikinheimo
P., Tuominen V., Ahonen A.-K., Teplyakov A., Cooperman B.S., Baykov A.A., Lahti
R., Goldman A.
Proc.
Natl. Acad. Sci. USA 98 (2001) 3121-3126
The wealth of kinetic and structural information
makes inorganic pyrophosphatases (PPases) a good model system to study the
details of enzymatic phosphoryl transfer. The enzyme accelerates
metal-complexed phosphoryl transfer 1010-fold: but how? Our structures of the
yeast PPase product complex at 1.15 A and fluoride-inhibited complex at
1.9 A visualize the active site in three different states:
substrate-bound, immediate product bound, and relaxed product bound. These span
the steps around chemical catalysis and provide strong evidence that a water
molecule (Onu) directly attacks PPi with a pKa vastly lowered by
coordination to two metal ions and D117. They also suggest that a low-barrier
hydrogen bond (LBHB) forms between D117 and Onu, in part because of steric
crowding by W100 and N116. Direct visualization of the double bonds on the
phosphates appears possible. The flexible side chains at the top of the active
site absorb the motion involved in the reaction, which may help accelerate
catalysis. Relaxation of the product allows a new nucleophile to be generated
and creates symmetry in the elementary catalytic steps on the enzyme. We are
thus moving closer to understanding phosphoryl transfer in PPases at the
quantum mechanical level. Ultra-high resolution structures can thus tease out
overlapping complexes and so are as relevant to discussion of enzyme mechanism
as structures produced by time-resolved crystallography.
Samygina
V.R., Popov, A.N., Rodina E.V., Vorobyeva N.N., Lamzin V.S., Polyakov K.M.,
Kurilova S.A., Nazarova T.I., Avaeva S.M.
Journal
of Molecular Biology 314 (2001) 633-645
Two structures of Escherichia coli
soluble inorganic pyrophosphatase (EPPase) complexed with calcium pyrophosphate
(CaPPi-EPPase) and with Ca2+ (Ca2+-EPPase)
have been solved at 1.2 and 1.1 ? resolution, respectively. In the presence of
Mg2+, this enzyme cleaves pyrophosphate (PPi) into two
molecules of orthophosphate (Pi). This work has enabled us to locate PPi
in the active site of the inorganic pyrophosphatases family in the presence of
Ca2+, which is an inhibitor of EPPase.
Upon PPi binding, two Ca2+
at M1 and M2 subsites move closer together and one of the liganded water
molecules becomes bridging. The mutual location of PPi and the
bridging water molecule in the presence of inhibitor cation is catalytically
incompetent. To make a favourable PPi attack by this water molecule,
modelling of a possible hydrolysable conformation of PPi in the CaPPi-EPPase
active site has been performed. The reasons for Ca2+ being the
strong PPase inhibitor and the role in catalysis of each of four metal ions are
the mechanistic aspects discussed on the basis of the structures described.
Bazhin
A.V., Slepova O.S., Tikhomirova N.K.
Bulletin
of Experimental Biology and Medicine 131 (2001) 350-352.
Serious changes in the retina, diagnosed as
retinal degeneration and uveitis, are observed only in the presence of high
titers of antibodies to recoverin (Ca2+-binding protein, a
paraneoplastic antigen) in the blood of rabbits. Negligible changes in the
retina of rabbits with low antibody titers are detected only by
cytohistochemical analysis of the retina. No changes in the retina develop in
rabbits intravenously injected with antibodies to recoverin.
Kazakov
S.V., Muronetz V.I., Dainiak M.B., Izumrudov V.A., Galaev I.Yu., Mattiasson B.
Macromol.
Biosci. 1 (2001) 157-163.
The effect of the conformational state of the
polymer coil on the properties of protein-polymer conjugates has been studied
for the conjugates of antibody (monoclonal antibody from 6C5 clone against
inactivated rabbit muscle glyceraldehyde-3-phosphate dehydrogenase; Ab) with
poly(methacrylic acid) (PMAA) or poly-(acrylic acid) (PAA). The pH-dependencies
of molecular properties and structural parameters of aqueous solutions (radius
of gyration, intensity of scattered light, hydrodynamic diameter, and
polydispersity index) of Ab, PMAA, and PAA and their conjugates, i. e., Ab-PMAA
and Ab-PAA, have been studied using static and dynamic light scattering
techniques. While free Ab aggregates in solution and precipitates at its
isoelectric point, the covalent attachment of a charged polymer to Ab prevents
its association and shifts the precipitation point towards more acidic values
(from pH 5.95 for Ab to pHl4.8 for Ab-PMAA). The predominant role of the
conformational status of the polymer in the process of conjugate precipitation
has been considered. Contrary to the precipitation of Ab-PMAA, the formation of
stable colloidal particles was suggested for Ab-PAA at pH a 4.8. In the
conjugates, polymer chains surround the protein globule in an extremely compact
manner while Ab significantly affects the polymer conformation. The essentially
larger hydrodynamic radii of conjugates, when compared with their radii of
gyration, confirm the strong interaction of conjugates with solvent molecules.
Nagradova
N.K.
FEBS
Letters 487 (2001) 327-332.
Interdomain interactions play an important role
in the structural organization of many enzymes and the conformational
flexibility of their molecules. In this review, the role of intrasubunit and
intersubunit domain-domain interactions in the origins of pre-existent
asymmetry of homo-oligomeric D-glyceraldehyde-3-phosphate dehydrogenase and
tryptophanyl-tRNA synthetase is discussed on the basis of recent X-ray data and
other available information about the properties of these and related enzymes.
In addition, a novel key function of interdomain interactions is considered:
their potential contribution to intramolecular channeling of intermediates
between active centers located on different subunits of a hetero-oligomeric
enzyme (a,b-heterodimeric carbamoyl phosphate synthetase).
Merckel M.C., Fabrichniy
I.P., Salminen A., Kalkkinen N., Baykov A.A., Lahti R., Goldman A.
Structure (Camb) 9 (2001)
289-297.
Streptococcus
mutans
pyrophosphatase (Sm-PPase) is a member of a relatively uncommon but widely
dispersed sequence family (family II) of inorganic pyrophosphatases. A
structure will answer two main questions: is it structurally similar to the
family I PPases, and is the mechanism similar? RESULTS: The first family II
PPase structure, that of homodimeric Sm-PPase complexed with metal and sulfate
ions, has been solved by X-ray crystallography at 2.2 A resolution. The
tertiary fold of Sm-PPase consists of a 189 residue a/b N-terminal domain and a 114
residue mixed b sheet C-terminal domain and bears
no resemblance to family I PPase, even though the arrangement of active site
ligands and the residues that bind them shows significant similarity. The
preference for Mn2+ over Mg2+ in family II PPases is
explained by the histidine ligands and bidentate carboxylate coordination. The
active site is located at the domain interface. The C-terminal domain is hinged
to the N-terminal domain and exists in both closed and open conformations.
CONCLUSIONS: The active site similiarities, including a water coordinated to
two metal ions, suggest that the family II PPase mechanism is
"analogous" (not "homologous") to that of family I PPases.
This is a remarkable example of convergent evolution. The large change in C-terminal
conformation suggests that domain closure might be the mechanism by which
Sm-PPase achieves specificity for pyrophosphate over other polyphosphates.
Shizawa N.,
Uchiumi T., Taguchi J., Kisseleva N.A., Baykov A.A., Lahti R., Hachimori A.
European Journal of Biochemistry / FEBS 268 (2001) 5771-5775.
The
sequence SRKKQxxP near the C-terminus is conserved in pyrophosphatases of the
recently discovered family II and includes a triplet of positively charged
residues, two of which (Arg295 and Lys296 in Bacillus subtilis
pyrophosphatase) are part of the active site and one (Lys297) is not. The
importance of this triplet for catalysis by B. subtilis pyrophosphatase has
been estimated by mutational analysis. R295K and K296R substitutions were found
to decrease the catalytic constant 650- and 280-fold, respectively, and
decrease the pKa of the essential acidic group by 1.1 and 0.5,
respectively. K297R substitution was found to increase the catalytic constant
4.7-fold and to markedly change the protein circular dichroism spectrum in the
range 250-300 nm. These results, together with the results of theoretical
modelling of the enzyme-substrate complex, provide support for the direct
involvement of Arg295 and Lys296 in substrate binding in family II
pyrophosphatases.
Highly
efficient and enantioselective enzymatic acylation of amines in aqueous medium
Guranda
D.T., van Langen L.M., van Rantwijk F., Sheldonb R.A., Svedas V.K.
Tetrahedron:
Asymmetry 12 (2001) 1645-1650
A new strategy based on the unique catalytic
properties, stability and enantioselectivity of the relatively unknown
penicillin acylase from Alcaligenes faecalis has been developed for the
effective and enantioselective acylation of amines in aqueous medium. In
contrast to lipase-catalyzed acylations in organic solvents, the penicillin
acylase-catalyzed acylation of amines in aqueous solution is a rapid and
chemoselective process leading to a product which can subsequently be
deacylated by the same enzyme, imposing secondary enantiocontrol and leading to
effective resolution.