I. Bioenergetics and photosynthesis

II. Cell biology and physiology

III. Enzymology

IV. Gene structure, regulation and evolution

V. Molecular virology

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The effects of transient pore opening on generation of the transmembrane gradient of electrical potential across the inner mitochondrial membrane (delta psi) induced by NADH oxidation through the external pathway as well as on the uncoupling effect of fatty acids were studied. The pore opening was monitored by changes in the delta psi value. The cycle of pore opening/closing was found to have only an insignificant effect on the sensitivity of delta psi to fatty acid uncoupling. Once this cycle is over, NADH oxidation in the presence of exogenous cytochrome c results in generation of delta psi. In the absence of cytochrome c, the generation of delta psi induced by oxidation of exogenous NADH is observed if the incubation medium pH has been decreased from 7.4 to 7.0. The generation of delta psi was inhibited by cyclosporin A. In isotonic salt medium containing 125 mM KCl, the maximum level of delta psi generated by exogenous NADH after the cycle of pore opening/closing was significantly lower than the maximum level of delta psi generated in hypotonic incubation medium. The data obtained in this work suggest that the cycle of pore opening/closing has little if any effect on the energy coupling in liver mitochondria, whereas the external pathway of NADH oxidation activated by this cycle may support the energy-dependent functions of liver mitochondria.
 
 

We show that Ca²⁺ loading of mitochondria substantially augments the myristate-induced decrease in the transmembrane electric potential difference (delta psi). 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-induced delta psi 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 carboxy-atractylate (CAtr) after myristate induces the CsA-sensitive uncoupling, but when added after myristate and CsA, CAtr produces a decrease in delta psi, 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.
 
 

Cytochrome bd is one of the two terminal quinol oxidases in the respiratory chain of Escherichia coli. The enzyme catalyzes charge separation across the bacterial membrane during the oxidation of quinols by dioxygen but does not pump protons. In this work, the reaction of cytochrome bd with O₂ and related reactions has been studied by time-resolved spectrophotometric and electrometric methods. Oxidation of the fully reduced enzyme by oxygen is accompanied by rapid generation of membrane potential (delta psi, negative inside the vesicles) that can be described by a two-step sequence of (i) an initial oxygen concentration-dependent, electrically silent, process (lag phase) corresponding to the formation of a ferrous oxy compound of heme d and (ii) a subsequent monoexponential electrogenic phase with a time constant <60 microseconds that matches the formation of ferryl-oxo heme d, the product of the reaction of O₂ with the 3-electron reduced enzyme. No evidence for generation of an intermediate analogous to the "peroxy" species of heme-copper oxidases could be obtained in either electrometric or spectrophotometric measurements of cytochrome bd oxidation or in a spectrophotometric study of the reaction of H₂O₂ with the oxidized enzyme. Backflow of electrons upon flash photolysis of the singly reduced CO complex of cytochrome bd leads to transient generation of delta psi the opposite polarity (positive inside the vesicles) concurrent with electron flow from heme d to heme b558 and backward. The amplitude of the delta psi produced by the backflow process, when normalized to the reaction yield, is close to that observed in the direct reaction during the reaction of fully reduced cytochrome bd with O₂ and is apparently associated with full transmembrane translocation of approximately one charge.
 
 

The possible mechanisms of electrogenic processes accompanying proton transport in bacteriorhodopsin are discussed on the basis of recent structural data of the protein. Apparent inconsistencies between experimental data and their interpretation are considered. Special emphasis is placed on the protein conformational changes accompanying the reprotonation of chromophore and proton uptake stage in the bacteriorhodopsin photocycle reserved.
 
 

Functioning of membrane proteins, in particular ionic channels, can be modulated by alteration of their arrangement in membranes. We addressed this issue by studying the effect of different chain length polylysines on the kinetics of ionic channels formed in a bilayer lipid membrane (BLM) by 0-pyromellitylgramicidin carrying three negative charges at the C-terminus. The method of sensitized photoinactivation was applied to the analysis of the channel association-dissociation kinetics (characterized by the exponential factor of the curve describing the time course of the flash-induced decrease in the transmembrane current, tau). Addition of polylysine to the bathing solutions of BLM led to the deceleration of the photoinactivation kinetics, i.e. to the increase in tau. It was shown here that for a series of polylysines differing in their chain lengths, the value of tau grew as their concentration increased above a threshold level until at a certain concentration of each polylysine tau reached maximum. At higher polylysine concentrations tau began to decrease and finally became close to the control level observed in the absence of polylysine. With lengthening of the polylysine chain the maximum value of tau increased, the concentration dependence became steeper, and the threshold concentration decreased. The increase in the ionic strength of the medium shifted the concentration dependence of tau to higher polylysine concentrations and decreased the maximum value of tau. It was concluded that the increase in tau was caused by the formation of domains of 0-pyromellitylgramicidin molecules induced by binding of polylysines. This can be related to functional aspects of polycation-induced sequestering of negatively charged transmembrane peptides in neutral membranes.
 
 

A Haemophilus influenzae gene encoding a protein with high homology to ArcB receptor protein from Escherichia coli has been cloned. An error in the previously reported sequence of this gene has been found, thus increasing its open reading frame. The cloned gene comprising the entire open reading frame restores oxygen-dependent regulation of succinate dehydrogenase in an ArcB-deficient E. coli strain. Thus, this gene is a functional analog of ArcB from E. coli. By screening partially sequenced bacterial genomes using the BLAST program, proteins with high homology to ArcB protein from E. coli were found in Salmonella typhi, Yersinia pestis, Vibrio cholerae, and Pasteurella multocida. Comparison of these proteins with ArcB protein from E. coli and H. influenzae revealed conserved amino acid regions. Transmembrane helix II was shown to be highly homologous in all the ArcB-type proteins. The involvement of this region in ArcB-mediated oxygen-dependent regulation is suggested.
 
 

In this paper we explain the wavelength-dependent oscillatory features in the pump-probe kinetics of the core LH1 antenna of Rhodopseudomonas viridis (Monshouwer, R.; Baltuška, A.; van Mourik, F.; van Grondelle, R. Time-resolved absorption difference spectroscopy of the LH1 antenna of Rhodopseudomonas viridis. J. Phys. Chem. A 1998, 102, 4360). A quantitative fit of the data was obtained using the doorway-window representation of the nonlinear optical response in the vibrational eigenstate basis. In contrast to LH1/LH2 complexes from the BChl a-containing species, the LH1 antenna of Rps. viridis is characterized by a strong coupling of the excitonic states with two underdamped low-frequency modes (58 and 110 cm^-1 at 77K). Following a short femtosecond excitation pulse, this gives rise to the intense oscillations observed in the pump-probe traces, including their time and excitation/detection wavelength dependence. Furthermore, it leads to a pronounced and specific heterogeneity of the major absorption band due to the combined effects of the exciton splitting, disorder and the presence of vibrational sidebands. The sharp maxima in the second derivative of the low temperature absorption spectrum (Monshouwer, R.; Visschers, R. W.; van Mourik, F.; Freiberg, A.; van Grondelle, R. Low-temperature absorption and site-selected fluorescence of the light-harvesting antenna of Rhodopseudomonas viridis. Evidence for heterogeneity. Biochim. Biophys. Acta, 1995, 1229, 373) were assigned to the lowest exciton-vibrational transitions. The wavelength dependence of the experimentally observed oscillatory pattern suggests a different vibrational coherence decay for the ground- and excited-state wavepackets. This can be explained by assuming the (incoherent) migration of the delocalized exciton (polaron) around the ring-like antenna with a characteristic time constant of 0.9-1.5 ps at 77K.
 
 

The absorption and circular dichroism spectra of the B800-850 complex from Chromatium minutissimum before and after the Triton X-100 treatment were simulated by means of standard exciton theory, taking in account inhomogeneous broadening. To explain the spectral changes of the B800-850 complex treated with Triton X-100, we have assumed that all bacteriochlorophyll pigments absorbing at 850 nm exhibit the same additional rotation of ~20^o around the axis perpendicular to the membrane plane. This has been sufficient to fit the transformation in absorption and circular dichroism spectra induced by detergent treatment of the B800-850 complex.
 
 

Membrane protein functioning basically depends on the supramolecular structure of the proteins which can be modulated by specific interactions with external ligands. The effect of a water-soluble protein bearing specific binding sites on the kinetics of ionic channels formed by gramicidin A (gA) in planar bilayer lipid membranes (BLM) has been studied using three independent approaches: (1) sensitized photoinactivation, (2) single-channel, and (3) autocorrelation measurements of current fluctuations. As shown previously [Rokitskaya, T. I., et al. (1996) Biochim. Biophys. Acta 1275, 221], the time course of the flash-induced current decrease in most cases follows a single-exponential decay with an exponential factor (tau) that corresponds to the gA single-channel lifetime. Addition of avidin does not affect tau for gA channels, but causes a dramatic increase in tau for channels formed by gA5XB, a biotinylated analogue of gA. This effect is reversed by addition of an excess of biotin to the bathing solution. The average single-channel duration of gA5XB was about 3.6 s as revealed by single-channel recording of the BLM current. After prolonged incubation with avidin, a long-lasting open state of the gA5XB channel appeared which did not close for more than 10 min. The data on gA5XB photoinactivation kinetics and single-channel measurements were confirmed by analysis of the corresponding power spectra of the current fluctuations obtained in the control, in the presence of avidin, and after the addition of biotin. We infer that avidin produces a deceleration of gA5XB channel kinetics by motional restriction of gA5XB monomers and dimers upon the formation of avidin and gA5XB complexes, which would stabilize the channel state and thus increase the single-channel lifetime.
 
 

The photodynamic activity of sulfonated aluminum phthalocyanines (AlPcSn, 1 £ n £  4) was found to correlate with their affinity for membrane lipids. Adsorbing to the surface of large unilamellar vesicles (LUVs), aluminum phthalocyanine disulfonate induced the highest changes in their electrophoretic mobility. AlPcS2 was also most efficient in mediating photoinactivation of gramicidin channels, as revealed by measurements of the electric current across planar lipid bilayers. The increase in the degree of sulfonation of phthalocyanine progressively reduced its affinity for the lipid bilayer as well as its potency of sensitizing gramicidin channel photoinactivation. The portion of photoinactivated gramicidin channels, alpha, increased with rising photosensitizer concentration up to some optimum. The concentration at which alpha was at half-maximum amounted to 80 nM, 30 nM, 200 nM, and 2 microM for AIPcSi, AlPcS1, AlPcS2, and AIPcS4, respectively. At high concentrations a was found to decrease, which was attributed to quenching of reactive oxygen species and self-quenching of the photosensitizer triplet state by its ground state. Fluoride anions were observed to inhibit both AlPcSn (2 £ n £ 4) binding to LUVs and sensitized photoinactivation of gramicidin channels. It is concluded that photosensitizer binding to membrane lipids is a prerequisite for the photodynamic motivation of gramicidin channels.
 
 

Uncoupling effects of laurate and lauryl sulfate have been studied in the isolated rat liver and skeletal muscle mitochondria. In the oligomycin-treated liver mitochondria, 0.02 mM laurate or 0.16 mM lauryl sulfate caused a two-fold stimulation of respiration, accompanied by a membrane potential decrease. Carboxyatractylate (CAtr) and glutamate (or aspartate) strongly decrease the effect of laurate and lauryl sulfate on respiratory rate and membrane potential (the recoupling effect). With both uncouplers, this effect is maximal for CAtr and glutamate (aspartate) at pH 7.8 and 7.0, respectively. Tetraphenyl phosphonium cations, which decrease negative membrane charges, cause an alkaline shift of these pH dependencies. Small amounts of lauryl sulfate, which increase the membrane negative charge, induce the opposite shift when laurate is used as an uncoupler. ADP, but not GDP, partially recouple with both laurate and lauryl sulfate. We conclude that lauryl sulfate-induced uncoupling in rat liver, like the uncoupling induced by laurate, is mediated by the ATP/ ADP and glutamate/aspartate antiporters. In skeletal muscle mitochondria uncoupled by laurate, 200 microM GDP causes partial recoupling which can be enhanced by a subsequent additions of CAtr, glutamate and serum albumin. CAtr added before GDP promotes a larger recoupling than when added after GDP and prevents the subsequent effect of GDP. ADP is effective as recoupler at lower concentrations that GDP, whereas CDP is without influence. Lauryl sulfate uncoupling of skeletal muscle mitochondria is GDP-resistant but is sensitive to ADP, CAtr, glutamate and serum albumin. Our data suggest that in skeletal muscle mitochondria a GDP-sensitive mechanism is involved in uncoupling induced by laurate. This mechanism is absent in liver mitochondria. Possible mechanisms of laurate and lauryl sulfate-induced uncoupling are discussed.
 
 

The water conductivity of desformylgramicidin exceeds the permeability of gramicidin A by two orders of magnitude. With respect to its single channel hydraulic permeability coefficient of 1.1 x 10^-12 cm³ s^-1, desformylgramicidin may serve as a model for extremely permeable aquaporin water channel proteins (AQP4 and AQPZ). This osmotic permeability exceeds the conductivity that is predicted by the theory of single-file transport. It was derived from the concentration distributions of both pore-impermeable and -permeable cations that were simultaneously measured by double barreled microelectrodes in the immediate vicinity of a planar bilayer. From solvent drag experiments, approximately five water molecules were found to be transported by a single-file process along with one ion through the channel. The single channel proton, potassium, and sodium conductivities were determined to be equal to 17 pS (pH 2.5), 7 and 3 pS, respectively. Under any conditions, the desformyl-channel remains at least 10 times longer in its open state than gramicidin A.
 
 

Interruption of the menA or menB gene in Synechocystis sp. PCC 6803 results in the incorporation of a foreign quinone, termed Q, into the A1 site of photosystem I with a number of experimental indicators identifying Q as plastoquinone-9. A global multiexponential analysis of time-resolved optical spectra in the blue region shows the following three kinetic components: 1) a 3-ms lifetime in the absence of methyl viologen that represents charge recombination between P700⁺ and an FeS- cluster; 2) a 750-microsecond lifetime that represents electron donation from an FeS- cluster to methyl viologen; and 3) an ~15-microsecond lifetime that represents an electrochromic shift of a carotenoid pigment. Room temperature direct detection transient EPR studies of forward electron transfer show a spectrum of P700⁺ Q^- during the lifetime of the spin polarization and give no evidence of a significant population of P700⁺ FeS- for t £ 2-3 microseconds. The UV difference spectrum measured 5 microseconds after a flash shows a maximum at 315 nm, a crossover at 280 nm, and a minimum at 255 nm as well as a shoulder at 290-295 nm, all of which are characteristic of the plastoquinone-9 anion radical. Kinetic measurements that monitor Q at 315 nm show a major phase of forward electron transfer to the FeS clusters with a lifetime of ~15 microseconds, which matches the electrochromic shift at 485 nm of the carotenoid, as well as an minor phase with a lifetime of ~250 microseconds. Electrometric measurements show similar biphasic kinetics. The slower kinetic phase can be detected using time-resolved EPR spectroscopy and has a spectrum characteristic of a semiquinone anion radical. We estimate the redox potential of plastoquinone-9 in the A1 site to be more oxidizing than phylloquinone so that electron transfer from Q- to Fx is thermodynamically unfavorable in the menA and menB mutants.
 
 

Recently knockout of the gene encoding an adaptor protein (p66^shc) was shown both to prolong the life span of an animal and to prevent apoptosis of cells in response to added H₂O₂ (Migliaccio et al. [1999] Nature 402, 309-313). A hypothesis is put forward in which p66shc is assumed to be involved in phenoptosis, i.e., programmed death of an organism, mediated by the reactive oxygen species-dependent massive apoptosis in an organ of vital importance. The reactive oxygen species are suggested to oxidize phosphatidyl serine in the inner leaflet of the cell plasma membrane, resulting in appearance of this phospholipid in the outer membrane leaflet, an effect recognized by a special receptor and causing the p66shc phosphorylation at a serine residue. Serine-phosphorylated p66shc there is proposed to block mitosis and initiate apoptosis. The large-scale apoptosis leads to phenoptosis and, hence, shortens the life span of the organism.
 
 

The very fact that mitochondria participate in amplification of the cell suicide signals has stimulated interest in the mechanism of this and related phenomena. It seems probable that mitochondria possess an autonomic system that allows them to commit suicide. This mitoptosis is mediated by reactive oxygen species (ROS), causing opening of the permeability transition pores (PTP) in the inner mitochondrial membrane. Mitoptosis can purify the mitochondrial population in a cell from the ROS-overproducing organelles. Massive mitoptosis can result in apoptosis (programmed cell death) because of the release of proapoptotic proteins from the mitochondrial intermembrane space, a mechanism purifying tissues from the ROS-overproducing and other unwanted cells. Large-scale apoptosis can be used by organisms to eliminate some organs during ontogenesis (organoptosis). In adult organisms, organoptosis of organs of vital importance may entail a programmed death of individuals (phenoptosis). This mechanism might purify kins, communities, and populations from individuals becoming dangerous because of, for example, heavy infection (septic shock). It is hypothesized that aging represents a slow ROS-linked phenoptosis that eliminates individuals with damaged genomes and gives reproductive advantage to those who succeeded in a better preservation of their genomes from damage.
 
 

Interaction of the two high-spin hemes in the oxygen reduction site of the bd-type quinol oxidase from Escherichia coli has been studied by femtosecond multicolor transient absorption spectroscopy. The previously unidentified Soret band of ferrous heme b595 was determined to be centered around 440 nm by selective excitation of the fully reduced unliganded or CO-bound cytochrome bd in the alpha-band of heme b595. The redox state of the b-type hemes strongly affects both the line shape and the kinetics of the absorption changes induced by photodissociation of CO from heme d. In the reduced enzyme, CO photodissociation from heme d perturbs the spectrum of ferrous cytochrome b595 within a few ps, pointing to a direct interaction between hemes b595 and d. Whereas in the reduced enzyme no heme d-CO geminate recombination is observed, in the mixed-valence CO-liganded complex with heme b595 initially oxidized, a significant part of photodissociated CO does not leave the protein and recombines with heme d within a few hundred ps. This caging effect may indicate that ferrous heme b595 provides a transient binding site for carbon monoxide within one of the routes by which the dissociated ligand leaves the protein. Taken together, the data indicate physical proximity of the hemes d and b595 and corroborate the possibility of a functional cooperation between the two hemes in the dioxygen-reducing center of cytochrome bd.
 
 

The excitation of bacterial reaction centers (RCs) at 870 nm by 30 fs pulses induces the nuclear wavepacket motions on the potential energy surface of the primary electron donor excited state P, which lead to the fs oscillations in stimulated emission from P |M.H. Vos. M.R. Jones, C.N. Hunter. J. Breton, J.-C. Lambry and J.-L. Martin (1994) Biochemistry 33, 6750-6757] and in Q_Y absorption band of the primary electron acceptor, bactcriochlorophyll monomer B_A |A.M. Streltsov, S.I.E. Vulto, A.Y. Shkuropatov, A.J. Hoff, T.J. Aartsma and V.A. Shuvalov (1998) J. Phys. Chem. B 102, 7293-7298| with a set of fundamental frequencies in the range of 10-300 cm^-1. We have found that in pheophytin-modified RCs, the fs oscillations with frequency around 130 cm^-1 observed in the P-stimulated emission as well as in the B_A absorption band at 800 nm are accompanied by remarkable and reversible formation of the 1020 nm absorption band which is characteristic of the radical anion band of bacteriochlorophyll monomer B_A^-. These results are discussed in terms of a reversible electron transfer between P and B_A induced by a motion of the wavepacket near the intersection of potential energy surfaces of P and P⁺B_A^-, when a maximal value of the Franck-Condon factor is created.
 
 

We sought to understand the relationship between reactive oxygen species (ROS) and the mitochondrial permeability transition (MPT) in cardiac myocytes based an the observation of increased ROS production at sites of spontaneously deenergized mitochondria. We devised a new model enabling incremental ROS accumulation in individual mitochondria in isolated cardiac myocytes via photoactivation of tetramethylrhodamine derivatives, which also served to report the mitochondrial transmembrane potential, delta psi. This ROS accumulation reproduce triggered abrupt (and sometimes reversible) mitochondrial depolarization. This phenomenon was ascribed to MPT induction because (a) bongkrekic acid prevented it and (b) mitochondria became permeable for calcein (~620 daltons) concurrently with depolarization. These photodynamically produced "triggering" ROS caused the MPT induction, as the ROS scavenger Trolox prevented it. The time required for triggering ROS to induce the MPT was dependent on intrinsic cellular ROS-scavenging redox mechanisms, particularly glutathione. MPT induction caused by triggering ROS coincided with a burst of mitochondrial ROS generation, as measured by dichlorofluorescein fluorescence, which we have termed mitochondrial "ROS-induced ROS release" (RIRR). This MPT induction/RIRR phenomenon in cardiac myocytes often occurred synchronously and reversibly among long chains of adjacent mitochondria demonstrating apparent cooperativity. The observed link between �РТ and RIRR could be a fundamental phenomenon in mitochondrial and cell biology.
 
 

The complete amino acid sequence of the protease inhibitor BWI-4a from buckwheat (Fagopyrum esculentum Moench) seeds has been established by automated Edman degradation in combination with MALDI-TOF mass spectrometry. The inhibitor molecule consists of 67 amino acid residues with a single disulfide bond. Its N-terminus is blocked by a pyroglutamic acid residue. The reactive site of the inhibitor contains an Arg43-Asp44 bond. Mass spectrometry revealed that inhibitor BWI-4a is present in buckwheat seeds in two isoforms differing by a single amino acid substitution of Gly40 for Ala40. Analysis of the amino acid sequence of the BWI-4a inhibitor indicates that this inhibitor is a member of the potato proteinase inhibitor I family.
 
 

The complete amino acid sequence of protease inhibitor BWI-4a from buckwheat (Fagopyruni esculentum Moench) seeds, consisting of 67 amino acid residues with a single disulfide bond, has been established by Edman degradation in combination with matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Its N terminus is blocked by a pyroglutamic acid residue. Mass spectrometric analysis revealed that inhibitor BWI-4a is present in buckwheat seeds in two isoforms with a single amino acid substitution of Ala⁴⁰ for Gly⁴⁰. The reactive site of the inhibitor contains an Arg⁴³-Asp⁴⁴ bond. Analysis of the amino acid sequence suggests that the buckwheat seed protease inhibitor is a member of the potato proteinase inhibitor I family.
 
 

In this paper, we describe the postural activity in sitting rats and rabbits. An animal was positioned on the platform that could be tilted in the frontal plane for up to ±20-30°, and postural corrections were video recorded. We found that in both rat and rabbit, the postural reactions led to stabilization of the dorsal-side-up trunk orientation. The result of this was that the trunk tilt constituted only -50% (rat) and 25% (rabbit) of the platform tilt. In addition, in the rabbit the head orientation was also stabilized. Trunk stabilization persisted in the animals subjected to the bilateral labyrinthectomy and blindfolding, suggesting that the somatosensory input is primarily responsible for trunk stabilization. Trunk stabilization was due to extension of the limbs on the side moving down, and flexion of the opposite limbs. EMG recordings showed that the limb extension was caused by the active contraction of extensor muscles. We argue that signals from the Golgi tendon organs of the extensor muscles may considerably contribute to elicitation of postural corrective responses to the lateral tilt.
 
 

Extracellular proteases secreted by the filamentous fungus Trichoderma harzianum have been identified. A proteinase active towards Z-Ala-Ala-Leu-pNa--the substrate of subtilisin-like proteases--dominated in the culture medium. This proteinase is synthesized de novo in response to addition of a protein substrate to the medium. Changing the carbohydrate in the culture medium changed the quantitative and qualitative spectrum of secreted enzymes. The most active extracellular proteinase of Trichoderma harzianum was purified 322-fold from the culture medium and obtained with a yield of 7.2%. The molecular mass of this proteinase is 73 kD and its pI is 5.35. The isolated enzyme has two distinct activity maxima, at pH 7.5 and 10.0, and is stable in the pH range 6.0-11.0. The temperature optimum for enzyme activity is 40°C at pH 8.0. The proteinase is stable up to 45-50°C (depending on the substrate used). Calcium ions stabilized the enzyme at 55-60°C. According to data on the study of functional groups of the active center and substrate specificity, the enzyme isolated from the culture medium of Trichoderma harzianum is a subtilisin-like serine proteinase.
 
 

Infection of cells with influenza virus is mediated by the virus envelope protein hem-agglutinin (HA) which induces fusion of viral and target membranes. Earlier we showed using fluorescent microscopy that HAb2 cells expressing HA on their plasma membranes fused with PLC cells when pH of the external medium was decreased to ~5. In the present work we used double whole-cell recording to monitor the intercellular conductance in HAb2/PLC cell pairs during fusion. In ~40% of cell pairs the pH drop induced the intercellular conductance, which we interpret as the formation of a fusion pore. The following stages of the conductance growth were distinguished: initial fluctuations near zero (flicker), a subsequent slow increase up to 1-4 nS and a final rapid increase up to 10-100 nS (complete fusion). The first detectable intercellular conductance change (opening of a fusion pore) was accompanied by an increase in the conductance of both HAb2 and PLC cell membrane. This observation suggests that the early pore complex should be leaky. The dynamics of the intercellular conductance appeared to depend upon the voltage difference between the fusing HAb2 and PLC cells: voltages higher than 40 mV facilitated the conductance growth.
 
 

The thermal stability of rabbit skeletal muscle glycogen phosphorylase b was characterized using enzymological inactivation studies, differential scanning calorimetry, and analytical ultracentrifugation. The results suggest that denaturation proceeds by the dissociative mechanism, i.e., it includes the step of reversible dissociation of the active dimer into inactive monomers and the following step of irreversible denaturation of the monomer. It was shown that glucose 1-phosphate (substrate), glucose (competitive inhibitor), AMP (allosteric activator), FMN, and glucose 6-phosphate (allosteric inhibitors) had a protective effect. Calorimetric study demonstrates that the cofactor of glycogen phosphorylase - pyridoxal 5'-phosphate - stabilizes the enzyme molecule. Partial reactivation of glycogen phosphorylase b preheated at 53°C occurs after cooling of the enzyme solution to 30°C. The fact that the rate of reactivation decreases with dilution of the enzyme solution indicates association of inactive monomers into active dimers during renaturation. The allosteric inhibitor FMN enhances the rate of phosphorylase b reactivation.
 
 

Differential scanning calorimetry (DSC) and light scattering were used to analyze the interaction of duck gizzard tropomyosin (tropomyosin) with rabbit skeletal-muscle F-actin. In the absence of F-actin. tropomvosin, represented mainly by heterodimers, unfolds at 41°C with a sharp thermal transition. Interaction of tropomvosin heterodimers with F-actin causes a 2-6°C shift in the tropomyosin thermal transition to higher temperature, depending on the tropomyosin/actin molar ratio and protein concentration. A pronounced shift of the tropomyosin thermal transition was observed only for tropomyosin heterodimers, and not for homodimers. The most pronounced effect was observed after complete saturation of F-actin with tropomyosin molecules, at tropomyosin/actin molar ratios > 1 : 7. Under these conditions, two well-separated peaks of tropomvosin were observed on the thermogram besides the peak of F-actin, the peak characteristic of free tropomyosin heterodimer, and the peak with a maximum at 45-47°C corresponding to tropomyosin bound to F-actin. By measuring the temperature-dependence of light scattering, we found that thermal unfolding of tropomyosin is accompanied by its dissociation from F-actin. Thermal unfolding of tropomyosin is almost completely reversible, whereas F-actin denatures irreversibly. The addition of tropomyosin has no effect on thermal unfolding of F-actin. which denatures with a maximum at 64°C in the absence and at 78°C in the presence of a twofold molar excess of phalloidin. After the F-actin-tropomyosin complex had been heated to 90°C and then cooled (i.e. after complete irreversible denaturation of F-actin). only the peak characteristic of free tropomyosin was observed on the thermogram during reheating, whereas the thermal transitions of F-actin and actin-hound tropomvosin completely disappeared. Therefore, the DSC method allows changes in thermal unfolding of tropomvosin resulting from its interaction with F-actin to be probed very precisely.
 
 

Interaction of isolated bacterial flagellum filaments (BFF) and intact flagella from E. coli MS 1350 and B. brevis G.-B.p⁺ with rabbit skeletal myosin was studied. BFF were shown to coprecipitate with myosin (but not with isolated myosin rod) at low ionic strength, that is, under conditions of myosin aggregation. The data of electron microscopy indicate that filaments of intact bacterial flagella interact with isolated myosin heads (myosin subfragment 1, S1), and this interaction is fully prevented by addition of Mg²⁺-ATP. Addition of BFF inhibited both K⁺-EDTA- and Ca²⁺-ATPase activity of skeletal muscle myosin, but had no effect on its Mg²⁺-ATPase activity. Monomeric flagellin did not coprecipitate with myosin and had no effect on its ATPase activities. BFF were shown to compete with F-actin in myosin binding. It is concluded that BFF interact with myosin heads and affect their ATPase activity. Thus, BFF composed of a single protein Hagellin are in many respects similar to actin filaments. Common origin of actin and flagellin may be a reason for this similarity.
 
 

Here. we demonstrate in a direct electrophysiological experiment that a neuron can form electrical connections to itself. An isolated identified neuron with a long axon was plated in culture and the axon was looped so that its distal end contacted the cell body. After two days in culture, the cell body and the axon were both impaled with microelectrodes and the axon segment between the recording electrodes was cut. Electrotonic coupling was revealed between the separated cell compartments immediately after axon transection. In contrast to an earlier publication [Guthrie P. B. et al. (1994) J. Neurosci. 14, 1477-1485], no constraints on the formation of the electrical connections between different parts of the same neuron were revealed in our experiments. Thus. these experiments demonstrate that in vitro culture of a single neuron can form reflexive electrical connections which may strongly affect the basic properties of the neuron and should be taken into account in both experimental and model electro-physiological studies.
 
 

In mammalian preovulatory oocytes, rRNA synthesis is down-regulated until egg fertilization and zygotic, genome reactivation, but the underlying regulatory mechanisms of this phenomenon are poorly characterized. We examined the molecular organization of the rRNA synthesis and processing machineries in fully grown mouse oocytes in relation to ongoing rDNA transcription and oocyte progression throughout meiosis. We show that, at the germinal vesicle stage, the two RNA polymerase I (RNA pol I) subunits, RPA116 and PAF53/RPA53, and the nucleolar upstream binding factor (UBF) remain present irrespective of ongoing rDNA transcription and colocalize in stoichiometric amounts within discrete foci at the periphery of the nucleolus-like bodies. These foci are spatially associated with the early pre-rRNA processing protein fibrillarin and in part with the pre-ribosome assembly factor B23/nucleophosmin, After germinal vesicle breakdown, the RNA pol I complex disassembles in a step-wise manner from chromosomes, while UBF remains associated 'with chromosopies until late prometaphase I. Dislodging of UBF, but not. of RNA pol I, is impaired by the phosphatase inhibitor okadaic acid, thus strengthening the idea of a relationship between UBF dynamics and protein phosphorylation. Since neither RNA pol I, UBF, fibrillarin, nor B23 is detected at metaphase II, i.e., the normal stage of fertilization, we conclude that these nucleolar proteins are not transported to fertilized eggs by maternal chromosomes. Together, these data demonstrate an essential difference in the dynamics of the major nucleolar proteins during mitosis and meiosis.
 
 

Recent progress in studies of the mode of action of cytoplasmic inorganic pyrophosphatases is mainly due to the analysis of a dozen and a half structures of the apoenzyme, its complexes, and mutants. However, despite considerable research on the mechanism of action of these enzymes, many important problems remain unclear. Among them is the problem of active site interactions in oligomeric structures and their role in catalysis; this review focuses on this problem. The abundant experimental data requires generalization and comprehensive analysis. A characteristic feature of the spatial structure of inorganic pyrophosphatases is a flexible system of noncovalent interactions between protein groups penetrating the whole molecule of the oligomeric enzyme. Binding of metal ions, sulfate (an analog of the product of the enzymatic reaction), and affinity phosphorus-containing inhibitors at the active site or site-directed mutagenesis induce rearrangements in the set of hydrogen and ionic interactions, which change active site properties and in some instances, cause molecule asymmetry. In the trimeric form of Escherichia coli pyrophosphatase obtained by dissociation of a hexamer, active sites also interact with each other, which is manifested by negative cooperativity upon substrate binding. The association of trimers into the hexamer leads to perfect organization of active sites and to their coordinated functioning, probably due to the restoration of communication channels between the trimers.
 
 

The causes of inhibition of Escherichia coli inorganic pyrophosphatase (PPase) by Ca²⁺ were investigated. The interactions of several mutant pyrophosphatases with Ca²⁺ in the absence of substrate were analyzed by equilibrium dialysis. The kinetics of Ca²⁺ inhibition of hydrolysis of the substrates MgPP_i and LaPP_i by the native PPase and three mutant enzymes (Asp-42-Asn, Ala, and Glu) were studied. X-Ray data on E. coli PPase complexed with Ca²⁺ or CaPP_i solved at atomic resolution were analyzed. It was shown that, in the course of the catalytic reaction, Ca²⁺ replaces Mg²⁺ at the M2 site, which shows higher affinity for Ca²⁺ than for Mg²⁺. Different properties of these cations account for active site deformation. Our findings indicate that the filling of the M2 site with Ca²⁺ is sufficient for PPase inhibition. This fact proves that Ca²⁺ is incapable of properly activating the H₂O molecule for nucleophilic attack on PP_i. It was also demonstrated that Ca²⁺, as a constituent of the non-hydrolyzable substrate analog CaPP_i, competes with MgPP_i at the M3 binding site. As a result, Ca²⁺ is a powerful inhibitor of all known PPases. Other possible reasons for the inhibitory effect of Ca²⁺ on the enzyme activity are also considered.
 
 

The fluoride ion is a potent and specific inhibitor of cytoplasmic pyrophosphatase (PPase). Fluoride action on yeast PPase during PP_i hydrolysis involves rapid and slow phases, the latter being only slowly reversible [Smirnova, I. N., and Baykov, A. A. (1983) Biokhimiya 48,1643-1653]. A similar behavior is observed during yeast PPase catalyzed PP_i synthesis. The amount of enzyme  PPi complex formed from solution P_i exhibits a rapid drop upon addition of fluoride, followed, at pH 7.2, by a slow increase to nearly 100% of the total enzyme. The slow reaction results in enzyme inactivation, which is not immediately reversed by dilution. These data show that fluoride binds to an enzyme  PP_i intermediate during the slow phase and to an enzyme  P_i intermediate during the rapid phase of the inhibition. In Escherichia coli PPase, the enzyme  PP_i intermediate binds F- rapidly, explaining the lack of time dependence in the inhibition of this enzyme. The enzyme  PP_i intermediate formed during PP_i hydrolysis binds fluoride much faster (yeast PPase) or tighter (E. coli PPase) than the similar complex existing at equilibrium with P_i. It is concluded that PPase catalysis involves two enzyme  PP_i intermediates, of which only one (immediately following PP_i addition and predominating at acidic pH) can bind fluoride. Simulation experiments have indicated that interconversion of the enzyme  PP_i intermediates is a partially rate-limiting step in the direction of hydrolysis and an exclusively rate-limiting step in the direction of synthesis.
 
 

Five catalytic functions of yeast inorganic pyrophosphatase were measured over wide pH ranges: steady-state PP_i hydrolysis (pH 4.8-10) and synthesis (6.3-9.3), phosphate-water oxygen exchange (pH 4.8-9.3), equilibrium formation of enzyme-bound PP_i (pH 4.8-9.3), and Mg²⁺ binding (pH 5.5-9.3). These data confirmed that enzyme-PP_i intermediate undergoes isomerization in the reaction cycle and allowed estimation of the microscopic rate constant for chemical bond breakage and the macroscopic rate constant for PP_i release. The isomerization was found to decrease the pK_a of the essential group in the enzyme-PP_i intermediate, presumably nucleophilic water, from >7 to 5.85. Protonation of the isomerized enzyme-PP_i intermediate decelerates PP_i hydrolysis but accelerates PP_i release by affecting the back isomerization. The binding of two Mg²⁺ ions to free enzyme requires about five basic groups with a mean pK_a of 6.3. An acidic group with a pK_a ~ 9 is modulatory in PP_i hydrolysis and metal ion binding, suggesting that this group maintains overall enzyme structure rather than being directly involved in catalysis.
 
 

Inhibition of penicillin acylases from Escherichia coli and Alcaligenes faecalis by aliphatic and aromatic alcohols was studied. It was shown that the inhibition of both enzymes has competitive nature and they bind the alcohols at the acyl group binding site of the enzyme active center. The free energy of alcohol sorption was shown to be linearly dependent on the hydrophobicity of the inhibitor with slopes of 1.6 and 1.7, demonstrating extremely effective hydrophobic interactions. To rationalize the observed distinctions in the inhibiting properties of aromatic and aliphatic alcohols beginning with butanol, it was suggested that the loss of entropy occurring on the interaction of the ligand with the tightly restricted hydrophobic pocket of the active center makes an essential contribution to the overall energetics of complex formation.
 
 

A new method for the production of monovalent Fab fragments of antibodies has been developed. Traditionally Fab fragments are produced by proteolytic digestion of antibodies in solution followed by isolation of Fab fragments. In the case of monoclonal antibodies against inactivated subunits of glyceraldehyde-3-phosphate dehydrogenase, digestion with papain resulted in significant damage of the binding sites of the Fab fragments. Antigen was covalently attached to the polycation, poly(N-ethyl-4-vinylpyridinium bromide). Proteolysis of monoclonal antibodies in the presence of the antigen-polycation conjugate followed by (i) precipitation induced by addition of polyanion, poly(methacrylic) acid, and pH shift from 7.3 to 6.5 and (ii) elution at pH 3.0 resulted in 90% immunologically competent Fab fragments. Moreover, the papain concentration required for proteolysis was 10 times less in the case of antibodies bound to the antigen-polycation conjugate than that of free antibodies in solution. The digestion of antibodies bound to the antigen-polyelectrolyte complex was less damaging, suggesting that binding to the antigen-polycation conjugate not only protected binding sites of monoclonal antibodies from proteolytic damage but also facilitated the proteolysis probably by exposing antibody molecules in a way convenient for proteolytic attack by papain.
 
 

This study describes the investigation of the possibility of genetic transformation of already immobilized competent cells by plasmids. The preliminary prepared competent cells were entrapped into granules of an insoluble carrier, a cryogel of poly (vinyl alcohol). The specific activity of organophosphatehydrolase and ampicillin resistance conferred by pOPfl plasmid were used as markers of successful transformation of the immobilized competent cells. The effect of main experimental conditions of transformation usually used for free cells, i. e., time of incubation of cells with DNA solution, temperature, and time of heat shock, on the transformation efficiency of the immobilized competent cells has been studied. A number of important factors of preparation of immobilized transformed cells, i. e., the concentration of immobilized competent cells inside the granules, the concentration of DNA solution used for transformation, have been shown to affect the OPH-activity of the final immobilized transformants. The possibility of transformation of the immobilized competent cells by both single- and double-stranded plasmid DNA molecules has been demonstrated.
 
 

The cell-to-cell movement of the GUS-tagged potato virus X (PVX) coat protein (CP) movement-deficient mutant was restored by potyviral CPs of potato virus A (PVA)-ahd potato virus Y (PVY) in Nicotians benthamiana leaves in transient cobombardment experiments. Viral cell-to-cell movement of PVX CP mutant was complemented in Nicotiana tabacum cv. SR1 transgenic plants expressing PVY CP: PVX RNA and polymerase were detected in the PVX CP mutant-inoculated leaves of transgenic plants. These findings demonstrated the ability of the PVX CP-deficient mutant to move from cell to cell but not long distances in the transgenic plants and suggest that CPs of potex- and potyviruses display complementary activities in the movement process. Potyviral CP alone is not able to carry out these activities, since the mutated PVX CP is indispensable for restored movement. No frans-encapsidation between potyviral CP and PVX RNA was observed. Therefore, potyviral CP facilitates the PVX CP mutant movement by the mechanism that cannot be explained by coat protein substitution. Our data also suggest that CP functioning in cell-to-cell movement is not restricted to a simple passive role in forming virions.
 
 

Data are presented concerning the possible participation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in regulation of the glycolytic pathway and the level of 2,3-diphosphoglycerate in erythrocytes. Experimental support has been obtained for the hypothesis according to which a mild oxidation of GAPDH must result in acceleration of glycolysis and in decrease in the level of 2,3-diphosphoglycerate due to the acyl phosphatase activity of the mildly oxidized enzyme. Incubation of erythrocytes in the presence of 1 mM hydrogen peroxide decreases 2,3-diphosphoglycerate concentration and causes accumulation of 3-phosphoglycerate. It is assumed that the acceleration of glycolysis in the presence of oxidative agents described previously by a number of authors could be attributed to the acyl phosphatase activity of GAPDH. A pH-dependent complexing of GAPDH and 3-phosphoglycerate kinase or 2,3-diphosphoglycerate mutase is found to determine the fate of 1,3-diphosphoglycerate that serves as a substrate for the synthesis of 2,3-diphosphoglycerate as well as for the 3-phosphoglycerate kinase reaction in glycolysis. A withdrawal of the two-enzyme complexes from the erythrocyte lysates using Sepharose-bound anti-GAPDH antibodies prevents the pH-dependent accumulation of the metabolites. The role of GAPDH in the regulation of glycolysis and the level of 2,3-diphosphoglycerate in erythrocytes is discussed.
 
 

Dynamics stimulation of the holotransketolase molecule revealed that the enzyme's conformation in crystal was different from that in solution. It was shown also that dissolved holotransketolase can bind aldose (the acceptor substrate) even in the absence of ketose (the donor substrate). The holotransketolase conformation did not change upon aldose binding unlike in the case of ketose binding/cleavage. Therefore the conformation of a catalytic complex of holotransketolase with an intermediate - i.e., a glycolaldehyde residue formed upon binding and subsequent cleavage of ketose - differed, at least in solution, from the conformation of both the free and aldose-complexed holotransketolase. Some structural peculiarities of the holotransketolase with the intermediate were established by means of molecular dynamics stimulation.
 
 

Recently we have initiated the use of synthetic polyelectrolytes to mimic the action of chaperones in living cells [Dainiak et al., Biochim. Biophys. Acta 1381 (1998) 279-285]. The next step in this direction is done by the synthesis of conjugates of poly(methacrylic acid) (PMAA) with antigen, denatured glyceraldehyde-3-phosphate dehydrogenase (dGAPDH), and with monoclonal antibodies specific for dGAPDH (but not for the native protein). The pH-dependent properties of the conjugates have been studied using turbidimetry and light scattering. The antibody-PMAA and dGAPDH-PMAA conjugates were shown to interact with free dGAPDH and antibodies respectively as well as with each other. Insoluble aggregates of dGAPDH with antibody-PMAA and of antibodies with dGAPDH-PMAA are formed in acidic media. The same situation occurs in the mixture of antibody-PMAA and dGAPDH-PMAA: precipitation takes place in acidic media, whereas soluble associates are formed in neutral solutions. The size of the soluble associates and the number of conjugates in the associate could be regulated by pH. The competition of free dGAPDH and dGAPDH-PMAA for binding with antibody-PMAA and the dynamic release of refolded GAPDH. with no affinity to antibody-PMAA, into solution could be used for simulating chaperone action.
 
 

A molecule of the photoreceptor Ca²⁺-binding protein recoverin contains four potential EF-hand Ca²⁺-binding sites, of which only two, the second and the third, are capable of binding calcium ions. We have studied the effects of substitutions in the second, third and fourth EF-hand sites of recoverin on its Ca²⁺-binding properties and some other characteristics, using intrinsic fluorescence, circular dichroism spectroscopy and differential scanning microcalorimetry. The interaction of the two operating binding sites of wild-type recoverin with calcium increases the protein's thermal stability, but makes the environment around the tryptophan residues more flexible. The amino acid substitution in the EF-hand 3 (E121Q) totally abolishes the high calcium affinity of recoverin, while the mutation in the EF-hand 2 (E85Q) causes only a moderate decrease in calcium binding. Based on this evidence, we suggest that the binding of calcium ions to recoverin is a sequential process with the EF-hand 3 being filled first. Estimation of Ca²⁺-binding constants according to the sequential binding scheme gave the values 3.7 x 10⁶ and 3.1 x 10⁵ M^-1 for third and second EF-hands, respectively. The substitutions in the EF-hand 2 or 3 (or in both the sites simultaneously) do not disturb significantly either tertiary or secondary structure of the apo-protein. Amino acid substitutions, which have been designed to restore the calcium affinity of the EF-hand 4 (G160D, K161E, K162N, D165G and K166Q), increase the calcium capacity and affinity of recoverin but also perturb the protein structure and decrease the thermostability of its apo-form.
 
 

Unlike wild type recoverin with only two (the second and the third) functioning Ca⁺²-binding sites out of four potential ones, the +EF4 mutant contains a third active Ca⁺²-binding site. This site was reconstructed from the fourth potential Ca⁺²-binding domain by the introduction of several amino acid substitutions in it by site-directed mutagenesis. The effect of these mutations in the fourth potential Ca⁺²-binding site of myristoylated recoverin on the structural features and conformational stability of the protein was studied by fluorimetry and circular dichroism. The apoform of the resulting mutant (free of Ca²⁺ ions) was shown to have a higher calcium capacity, significantly lower thermal stability, and noticeably different secondary and tertiary structures as compared with the apoform of wild-type recoverin.
 
 

Individual subunits of penicillin acylase from E. coli were isolated by gel-filtration under denaturing conditions (8 M urea). Recovery of the catalytic activity of the penicillin acylase heterodimer was studied after removal of urea. In the case of the heterodimer, 40-60% of the initial activity was recovered, whereas the activity of individual subunits was not recovered. Combination of native enzyme subunits with subunits isolated from the enzyme pre-inactivated with the irreversible inhibitor phenylmethylsulfonyl fluoride resulted in heterodimers which were active only in the case of involvement of the beta-subunit of the active enzyme.
 
 

Two substrates of the transketolase reaction are known to bind with the enzyme according to a ping-pong mechanism [1]. It is shown in this work that high concentrations of ribose-5-phosphate (acceptor substrate) compete with xylulose-5-phosphate (donor substrate), suppressing the transketolase activity (K_i = 3.8 mM).  However, interacting with the donor-substrate binding site on the protein molecule, the acceptor substrate, unlike the donor substrate, does not cause any change in the active site of the enzyme. The data are interesting in terms of studying the regulatory mechanism of the transketolase activity and the structure of the enzyme-substrate complex.
 
 

We present a new way of combined treatment of parodontitis, including consecutive applications of anti-inflammatory therapy and chitosan electrophoresis. Using this method we observe a considerable diminution of the depth of pathological gum pockets and the cessation of suppuration from them, as well as the disappearance of tooth mobility, restoration of blood circulation in tissues, normalization of chewing processes, and finally the complete rehabilitation of parodont functions. The clinical applications of chitosan during a 10 year period showed neither side effects nor contra-indications.
 
 

The structural properties of myristoylated forms of recombinant recoverin of the wild type and of its mutants with damaged second and/or third Ca²⁺-binding sites were studied by fluorimetry and circular dichroism. The interaction of wild-type recoverin with calcium ions was shown to induce unusual structural rearrangements in its molecule. In particular, protein binding with Ca²⁺ ions results in an increase in the mobility of the environment of Trp residues, in hydrophobicity, and in thermal stability (its thermal transition shifts by 15^oC to higher temperatures) but has almost no effect on its secondary structure. Similar structural changes induced by Ca²⁺ are also characteristic of the -EF2 mutant of recoverin whose second Ca²⁺-binding site is modified and cannot bind calcium ions. The structural properties of the -EF3 and -EF2,3 mutants (whose third or simultaneously second and third Ca²⁺-binding sites, respectively, are modified and damaged) are practically indifferent to the presence of calcium ions.
 
 

Penicillin acylase-catalyzed ampicillin synthesis via acyl group transfer in aqueous solution is highly dependent on the initial substrate concentration. The solubility of one substrate, 6-aininopenicillanic acid (6-APA), can be advantageously enhanced by the presence of acyl donor, the second substrate. Furthermore, a comparison of enzymatic synthesis in homogeneous solution with synthesis in a heterogeneous system having partially undissolved reactants, reveals major advantages for the latter approach. In this ''aqueous solution-precipitate" system, accumulation of both products, ampicillin and D-(^_)-phenylglycine, proceeds through the formation of their supersaturated solutions. Subsequent precipitation of the product ampicillin positively influences the efficiency of the biocatalytic process. As a result, ampicillin synthesis proceeds in 93% conversion on 6-APA and in 60'i'o conversion on D-(^_)-phenylglycine methyl ester.
 
 

Kinetic regularities of the enzymatic acyl group transfer reactions have been studied using ampicillin synthesis catalyzed by E. coli penicillin acylase as an example. It was shown that ampicillin synthesis proceeds through the formation of an acylenzyme-nucleophile complex capable of undergoing hydrolysis. The relative nucleophile reactivity of 6-aminopenicillanic acid (6-APA) is a complex parameter dependent on the nucleophile concentration. The kinetic analysis showed that the maximum yield of antibiotic being synthesized depended only on the nucleophile reactivity of 6-APA, the ratio between the enzyme reactivities with respect to the target product and acyl donor, and the initial concentrations of reagents. The parameters characterizing the nucleophile reactivity of 6-APA have been determined. The algorithm of modeling the enzymatic synthesis has been elaborated. The proposed algorithm allows the kinetics of the process not only in homogeneous, but also in heterogeneous ("aqueous solution-precipitate") systems to be quantitatively predicted and described based on experimental values of parameters of the reaction. It was shown that in heterogeneous "aqueous solution-precipitate" systems PA-catalyzed ampicillin synthesis proceeds much more efficiently compared to the homogeneous solution.
 
 

To obtain recombinant restriction endonuclease (R) and methylase (M) of the EcoRII restriction-modification system, bacterial strains overproducing their functional hexahistidine derivatives were constructed. Active full-length R·EcoRII was produced only in cells that also expressed M·EcoRII from a multi-copy plasmid. Recombinant R·EcoRII bound with hybrid DNA-RNA duplexes.
 
 

Subtilisin 72 suspension retained high activity and stability in the triple mixtures acetonitrile/DMF/water (DMF concentration was up to 70% (v/v)). The synthetic activity of subtilisin suspension was investigated using the model icadion of tetrapeptide Z-Ala-Ala-Leu-Phe-pNA. formation from Z-Ala-Ala-Leu-OMe and Phe-pNA ([S] = 30mM. [E] = 6 microM; [S]/[E] molar ratio 5000:1). In the systems containing up to 60% DMF the 95% product yield was reached within 2h. With DMF concentration increasing to 95%. the subtilisin catalytic efficiency notably decreased, though the product yield was still 30%. In the mixture acetonitrile /80% DMF/water, the effects of enzyme concentration, the reaction time and water content on the reaction progress were studied.
 
 

Results of a first successful application of a direct photo-induced affinity modification of Tet repressor (TetR (D)) protein with tetracycline within a complex of known three-dimensional structure are described. The conditions of the modification have provided suitable yields of the modified complex and allowed characterization of the modified segments of the protein. The potential of tetracycline as a fine modifying reagent was established. In the complex of TetR (D) protein with tetracycline, the antibiotic modifies at least two segments, Ile59-Glu73 and Ala173-Glu183, which form a binding tunnel for the drug according to the X-ray analysis. These data open possibilities for the use of different tetracycline targets for structural studies in solution.
 
 

A HeLa cell line expressing the green fluorescent protein fused to the SV40 T-antigen nuclear localization signal (EGFP-NLS) was established. Fluorescence in these cells was confined to the nuclei. After poliovirus infection, cytoplasmic fluorescence in a proportion of cells could be detected by 1 h postinfection (p.i.) and in virtually all of the fluorescent cells by 2 h p.i. The relocation could be prevented by cycloheximide but not by inhibition of poliovirus replication by guanidine^.HCI. Nuclear exit of a protein composed of three copies of GFP fused to the NLS also occurred upon poliovirus infection. A similar redistribution of EGFP-NLS took place upon infection with coxsakievirus B3 and, to a lesser extent, with vesicular stomatitis virus. The EGFP-NLS efflux was not due to the loss of NLS. Thus, some positive-strand and negative-strand RNA viruses trigger a rapid nonspecific relocation of nuclear proteins.
 
 

The divalent cation binding properties of human prothymosin alpha, an abundant nuclear protein involved in cell proliferation, were evaluated. By using prothymosin alpha retardation on a weak cation chelaling resin charged with various divalent cations, specific binding of Zn²⁺ ions by prothymosin alpha was observed. This finding was further confirmed by the equilibrium dialysis analysis which demonstrated that, within the micromolar range of Zn²⁺ concentrations, prothymosin alpha could bind up to three zinc ions in the presence of 100 mм NaCI and up to 13 zinc ions in the absence of NaCI. Equilibrium dialysis analysis also revealed that prothymosin alpha could bind Ca²⁺, although the parameters of Ca²⁺ binding by prothymosin alpha were less pronounced than those of Zn²⁺ binding in terms of the number of metal ions bound, the Ð�D values, and the resistance of the bound metal ions to 100 Ñ�м NaCI. The effects of Zn²⁺and Ca²⁺ on the interaction of prothymosin alpha with its putative partners, Rev of HIV type 1 and histone HI, were examined. We demonstrated that Rev binds prothymosin alpha, and that prothymosin alpha binding to Rev but not to histone HI was significantly enhanced in the presence of zinc and calcium ions. Our data suggest that the modes of prothymosin alpha interaction with Rev and histone HI are distinct and that the observed zinc and calcium-binding properties of prothymosin alpha might be functionally relevant.
 
 

We observed fragmentation of an essential proliferation-related human nuclear protein prothymosin alpha in the course of apoptosis induced by various stimuli. Prothymosin alpha cleavage occurred at the DDVD⁹⁹ motif. In vitro, prothymosin alpha could be cleaved at D⁹⁹ by caspase-3 and -7. Caspase hydrolysis disrupted the nuclear localization signal of prothymosin alpha and abrogated the ability of the truncated protein to accumulate inside the nucleus. Prothymosin alpha fragmentation may therefore be proposed to disable intranuclear proliferation-related function of prothymosin alpha in two ways: by cleaving off a short peptide containing important determinants, and by preventing active nuclear uptake of the truncated protein.
 
 

A series of fluorogenic tetra-, penta-, and hexapeptide substrates of the general structure Abz-X-Phe-Phe-Y-Ded or (-pNa in place of -Ded), where X = Ala, Ala-Ala, or Val-Ala and Y = -, Ala, or Ala-Ala, were proposed. Kinetic parameters of hydrolysis of these substrates by pepsin, cathepsin D, human gastricsin, pig pepsin, calf chymosin, and aspergillopepsin A were determined. The compounds synthesized proved to be effective substrates for aspartyl proteases of diverse origins.
 
 

High-molecular weight virus RNA on the surfaces of various substrates has been studied by atomic force microscopy (AFM). The process of releasing virus RNA from the protein shell under the action of chemical reagents has been studied. Special attention has been paid to the procedure of the preparation of samples and the immobilization of the structures under study. Images of the starting virus particles, partially deproteinized virus structures, and completely released RNA have been obtained. The images obtained have been statistically processed. The factors restricting the spatial resolution of the SFM patterns of the structures under study have been analysed.
 
 

The reversed-phase high-performance liquid chromatographic method employing photometry and fluorescence detection is described for the precise reproducible simultaneous measurement of total homocysteine (tHcy), cysteine (Cys), and glutathione (GSH) in human blood. Sample preparation involves conversion of disulfides to free thiols with tri-phenylphosphine, precipitation of proteins with trichloroacetic acid, conjugation of the thiols with monobromobimane (mBrB). The aminothiol assay is optimized by reduction and derivatization step conditions (pH, temperature and time of reactions) to obtain reliable quantitative results within the concentration range corresponding to normal and pathological levels of these thiols in human blood.
 
 

The fast isocratic and gradient reversed-phase high-performance liquid chromatographic methods employing photometric and/or fluorescence detection are 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. The aminothiol assay is optimized by reduction and derivatization step conditions (pH, temperature and time of reactions), as well as by chromatographic conditions to obtain reliable quantitative results within the concentration range corresponding to the levels of these thiols in human blood in norm and pathology. Its sensitivity allows the detection of aminothiol quantities >2 pmol.
 
 

A new high-performance capillary electrophoresis assay for aminothiols in human blood, including homocysteine, a marker of several human metabolism disorders, has been developed. Sample preparation involves conversion of disulfides to free thiols with triphenylphosphine, precipitation of proteins with sulfosalicylic acid, and conjugation of the thiols with monobromobimane. Derivatized thiols were separated in a sodium phosphate buffer using a fused-silica capillary (65 cm ´ 50 micro m I.D.) at 30°C. With the electric field of 250 V cm^-1, separation of homocysteine, glutathione and cysteine occurred at less than 10 min. Detection at 250 or 234 nm was used to confirm the monobimane-thiols peaks. The detection limit was ~5 nmol/ml for all labeled aminothiols. The proposed method for these compounds' analysis included simple sample preparation, high selectivity, good linearity (r²>0.999), high reproducibility (within-run precision for derivatized aminothiol peaks area RSD<5% for three times consequently injected sample); high reliability and the small volumes required for analysis made it suitable for clinical studies.
 
 

Chemical synthesis of 2'-O-(allyloxycarbonyl)methyladenosine, 2'-O-(methoxycarbonyl)methyladenosine and 2'-O-(2,3-dibenzoyloxy)propyluridine 3'-2-cyanoethyl-N,N-diisopropyl phosphoramidite building blocks are describes. These monomers were used successfully to incorporate carbxylic acid, 1,2-diol and aldehyde functionalities into synthetic oligonucleotides.
 
 

The subtilisin-sodium dodecyl sulfate complex was shown to catalyze the coupling of peptide segments on a solid phase in organic medium. By a two-stage enzymic condensation of peptide fragments on aminosilochrom (A) containing Met-Ala-Gly as a spacer, Dnp(or Boc)-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Met-Ala-Gly-A and Z-Ala-Ala-Glu(OMe)-Ala-Ala-Leu-Met-Ala-Gly-A were obtained. It was shown that the condensation products can be split off from the support using Met residue cleavage by BrCN.
 
 

A method of irrational oligonucleotide design, SELEX, is considered. Individual SELEX products, aptamers, are small molecules (40-100 nt) that have a unique three-dimensional structure, which provides for their specific and high-affinity binding to targets varying from low-molecular-weight ligands to proteins. Thus, the sophisticated biosynthesis of recognizing protein elements, antibodies, can be emulated in vitro via selection and synthesis of principally new recognizing elements based on nucleic acids.
 
 

It has earlier been shown that CYP11A1 (cytochrome P450scc precursor), synthesized in yeast cells, is imported into yeast mitochondria. However, in large part the foreign protein undergoes degradation or aggregates. In this work, we tried to prevent aggregation of CYP11A1 and stimulate its insertion into the mitochondrial inner membrane by substituting cholesterol (a substrate for cytochrome P450scc) for ergosterol in yeast cells. To this end, an ergosterol-deficient Saccharomyces cerevisiae mutant, growing in the presence of cholesterol and expressing a modified bovine CYP11A1 gene, was used. Under defined conditions, the mitochondrial respiratory system developed in this yeast and CYP11A1 with the CoxIV targeting presequence was imported into the mitochondria, being then proteolytically processed. However, substitution of cholesterol for ergosterol did not result in lowered aggregation of the imported CYP11A1 and its increased content in the SMP fraction. Hence, the presence of cholesterol is not instrumental in proper intramitochondrial compartmentalization and folding of CYP11A1.
 
 

A target sequence-specific DNA binding region of the restriction endonuclease Ssoll was identified by photocross-linking with an oligodeoxynucleotide duplex which was substituted with 5-iododeoxy-uridine (5-ldU) at the central position of the Ssoll recognition site (CCNGG). For this purpose the Ssoll-DNA complex was irradiated with a helium/cadmium laser (325 nm). The cross-linking yield obtained was -50%. In the presence of excess unmodified oligodeoxynucleotide or with oligodeoxynucleotides substituted with 5-ldU elsewhere, no cross-linking was observed, indicating the specificity of the cross-linking reaction. The cross-linked Ssoll-oligodeoxynucleotide complex was digested with chymotrypsin, a cross-linked peptide-oligodeoxynucleotide complex isolated and the site of cross-linking identified by Edman sequencing to be Trp61. In line with this identification is the finding that the W61A variant cannot be cross-linked with the IdU-substituted oligodeoxynucleotide, shows a decrease in affinity towards DNA and is inactive in cleavage. It is concluded that the region around Trp61 is involved in specific binding of Ssoll to its DNA substrate.
 
 

The aim of this work was to construct an anti-messenger targeted to the pim-\ oncogene transcript, based on circular or clamp oligodeoxyribonucleolides. The formation of bimolecular triplexes by clamp or circular oligonucleotides was investigated using single-stranded targets of both DNA (5'-CCCTCCTTTGAAGAA-3') and RNA type (5'-CCCUCCL'UUGAAGAA-3'). The third, 'Hoogsteen' strand of the triplex was represented by G,T-rich sequences. The secondary structures of the complexes were determined by thermal denaturation, circular dichroism and gel mobility shift experiments and shown to depend on the nature of the target strand. With DNA as target, the sequence of a clamp (or circular) oligonucleotide that formed the triple helix was 3'-GGGAGGAAACTTCTTTT-TTGTTGTTT-TT-GGTGGG-5', where the first TT dinucleotide (in italics) is a linker and the second TT (bold) represents the bridge through which the 'Hoogsteen' strand switches from one strand of the Watson-Crick duplex to the other, once the duplex is formed by the corresponding portion of the anti-messenger (underlined). The portion of the 'Hoogsteen' sequence of the triplex between the two TT dinucleotides binds to the 3' extremity of the target strand and runs parallel to it. The portion situated at the 5' end of the oligonucleotide switches to the purine tract of the complementary strand of the duplex and is antiparallel to it. In contrast, with RNA as target, for a branched clamp oligonucleotide that formed a triple helix over its entire length (5'-TTCTTCAAAGGAGGG-3'/\3>-GGGTGGTTT-T-GTTGTT-5') the portion of the 'Hoogsteen' sequence that bound to the 3' extremity of the target strand had to be antiparallel to it.
 
 

We have constructed plasmids for yeast expression of the fusion protein pre-cytochrome P450scc-adrenodoxin reductase-adrenodoxin (F2) and a variant of F2 with the yeast CoxIV targeting presequence. Mitochondria isolated from transformed yeast cells contained the F2 fusion protein at about 0.5% of total protein and showed cholesterol hydroxylase activity with 22(R)-hydroxycholesterol. The activity increased 17- or 25-fold when sonicated mitochondria were supplemented with an excess of purified P450scc or a mixture of adrenodoxin (Adx) and adrenodoxin reductase (AdxRed), respectively. These data suggest that, at least in yeast mitochondria, the interactions of the catalytic domains of P450scc, Adx, and AdxRed in the common polypeptide chain are restricted.
 
 

Cap-independent translation initiation on picornavirus mRNAs is mediated by an internal ribosomal entry site (IRES) in the 5' untranslated region (5' UTR) and requires both eukaryotic initiation factors (elFs) and IRES-specific cellular trans-acting factors (ITAFs). We show here that the requirements for trans-acting factors differ between related picornavirus IRESs and can account for cell type-specific differences in IRES function. The neurovirulence of Theiler's murine encephalomyelitis virus (TMEV; GDVII strain) was completely attenuated by substituting its IRES by that of foot-and-mouth disease virus (FMDV). Reconstitution of initiation using fully fractionated translation components indicated that 48S complex formation on both IRESs requires eIF2, eIF3, eIF4A, eIF4B, eIF4F, and the pyrimidine tract-binding protein (PTB) but that the FMDV IRES additionally requires ITAF₄₅, also known as murine proliferation-associated protein (Mpp1), a proliferation-dependent protein that is not expressed in murine brain cells. ITAF₄₅ did not influence assembly of 48S complexes on the TMEV IRES. Specific binding sites for ITAF₄₅, PTB, and a complex of the eIF4G and eIF4A subunits of eIF4F were mapped onto the FMDV IRES, and the cooperative function of PTB and ITAF4; in promoting stable binding of eIF4G/4A to the IRES was characterized by chemical and enzymatic footprinting. Our data indicate that PTB and ITAF₄₅ act as RNA chaperones that control the functional state of a particular IRES and that their cell-specific distribution may constitute a basis for cell-specific translational control of certain mRNAs.
 
 

Oligonucleotide inhibitors of the HIV-1 DNA integration identified to date are reviewed. Two basic strategies of blocking the integration are considered: shielding the integrase-binding sites on the viral DNA by triplex-forming oligonucleotides, and directly inhibiting the enzyme with oligonucleotide agents.
 
 

Contacts of phosphate groups at positions -12, -15, and -18 in relation to the transcription initiation site in the non-template strand of lac UV5 promoter with lysines or histidines of E. coli RNA polymerase in the open complex model were studied. A number of synthetic oligonucleotides from the -10-area of the non-template strand containing activated 5'-terminal phosphate group were cross-linked with holo- or core-enzyme of RNA polymerase. 5'-N-Hydroxybenzotriazole phosphodiesters of oligonucleotides were used as phosphate activated derivatives. They are capable of phosphorylating amino groups of lysines and histidines in the enzyme molecule that are brought into proximity with activated phosphate in the complex, resulting in the formation of a covalent bond between the oligonucleotide and the protein. The analysis of the products of cross-linking allowed the protein subunit and the amino acid residue taking part in the formation of the covalent bond for each oligonucleotide to be identified. It was found that all oligonucleotides from the non-template strand of promoter in the complex with the holo-enzyme are bound with the sigma70-subunit. When analyzing the products of partial cleavage of the complexes cross-linked at cysteines and methionines using SDS-PAGE, it was shown that phosphate at position -12 made contacts with His180 or His242 of the sigma70-subunit, the reactive amino acid residue being located between the first and second conservative regions. Phosphate at position -15 is located near lysines from two different areas--between Met413 and Met456 (regions 2.3 and 2.4) and between Met470 and Met507 (region 3.1). Phosphate at position -18 makes preferential contacts with a lysine situated between Met470 and Met507 (region 3.1). Based on the analysis of contacts of phosphate groups and the structure of the isolated sigma70-subunit established previously, a scheme of the mutual arrangement of the oligonucleotide and the sigma70-subunit possessed by the holo-enzyme has been proposed.
 
 

Homogeneous aminopeptidase PC was isolated with yield 67% and purification degree 237 from the hepatopancreas of the Kamchatka crab Paralithodes camtshatica by ion-exchange chromatography on DEAE-Sepharose, hydrophobic chromatography on Phenyl-Sepharose, and gel-filtration on Sephadex G-150. The enzyme is a homodimer with a molecular mass 220 kD (110 x 2). Aminopeptidase PC has pI = 4.1. It hydrolyzes Leu-pNA optimally at pH 6.0 and at the optimum temperature 36-40°C; in the presence of Ca²⁺ the enzyme is stable at pH 5.5-8.0. Aminopeptidase PC is activated by Ca²⁺, Mg²⁺, and Fe²⁺; it is completely inhibited by EDTA, o-phenanthroline, and bestatin. The enzyme contains four Zn atoms per molecule and is therefore a metalloaminopeptidase. The aminopeptidase PC can effectively cleave N-terminal Arg and Lys residues as well as Leu, Phe, and Met residues. K_m and k_cat values for hydrolysis of Leu-pNA were 0.075 mM and 0.19 sec^-1 and for hydrolysis of Arg-pNA 0.078 mM and 0.48 sec^-1, respectively. D-Amino acid residues cannot be cleaved. Thus, aminopeptidase PC of the Kamchatka crab has a mixed substrate specificity which is characteristic of some microbe aminopeptidases. Its N-terminal sequence ESVEIELPEGLSPLV is 46% coincident with that of yeast vacuolar aminopeptidase YSCA.
 
 

Alchemilla L. (Rosaceae) contains numerous agamospermous microspecies, which are often treated as species. However, many of them are not clearly morphologically distinct, and their genetic variability is practically not investigated. In the present study, we used RAPD analysis to assess the genetic relatedness between Alchemilla microspecies. In all, 51 plants from 12 Alchemilla microspecies were analysed, and 116 characters were considered (68 RAPD bands over three primers and 48 morphological characters). Phylogenetic trees were constructed by the unweighted pair-group method, neighbour-joining and maximum parsimony methods. The genetic data supported most Frohner's system of sections. Despite the use of a limited set of data in the investigation and weak support values, some tentative conclusions could be based on congruence of the RAPD analysis and morphological data. Alchemilla acutiloba Opiz and A. micans Buser should be united as a single microspecies. A. micans; section Plicutae should be divided into two series Pubescentes and Barbulutae, and A. heptagona Juz. may be separated in Exuentes series of Ultravulgares.
 
 

The proximity of loop D of 5 S rRNA to two regions of 23 S rRNA, domain II involved in translocation and domain V involved in peptide bond formation, is known from previous cross-linking experiments. Here, we have used site-directed mutagenesi.s and chemical probing to further define these contacts and possible sites of communication between 5 S and 23 S rRNA. Three different mutants were constructed at position A960, a highly conserved nucleotide in domain II previously crosslinked to 5 S rRNA, and the mutant rRNAs were expressed from plasmids as homogeneous populations of ribosomes in Eschericllia coli deficient in all seven chromosomal copies of the rRNA operon. Mutations A960U, A960G and, particularly, A960C caused structural rearrangements in the loop D of 5 S rRNA and in the peptidyltransferase region of domain V, as well as in the 960 loop itself. These observations support the proposal that loop D of 5 S rRNA participates in signal transmission between the ribosome centers responsible for peptide bond formation and translocation.
 
 

A proliferation-related human protein prothymosin alpha displays exclusively nuclear localization when produced in human and Saccharomyces cerevisiae cells, whereas its isolated bipartite NLS confers nuclear targeting of the GFP reporter in human but not in yeast cells. To test whether this observation is indicative of the existence of specific requirements for nuclear targeting of proteins in yeast, a set of prothymosin alpha deletion mutants was constructed. Subcellular localization of these mutants fused to GFP was determined in yeast and compared with their ability to bind yeast importin alpha (Srp1p) in vitro. The NLS of prothymosin alpha turned out to be both necessary and sufficient to provide protein recognition by importin alpha. However, the NLS-importin alpha interaction did not ensure nuclear targeting of prothymosin alpha - derivatives. This defect could be complemented by adding distinct prothymosin alpha sequences to the NLS-containing import substrate, possibly by providing binding site(s) for additional components of the yeast nuclear import machinery.
 
 

It was found that production of superoxide (O₂^·-) is crucial for normal morphogenesis of etiolated wheat seedlings in the early stages of plant development. The development of etiolated wheat seedlings was shown to be accompanied with cyclic changes in the rate of O₂^·- production both in the entire intact seedling and in its separated organs (leaf, coleoptile). First increase in the rate of O₂^·- production was clearly observed in the period from two to four days of seedling development, then the rate of O₂^·- production decreased to the initial level, and then it increased again for two days to a new maximum. An increase in O₂^·- production in the period of the first four days of seedling development correlates with an increase in DNA and protein contents in the coleoptile. The second peak of increased rate of O₂^·- production observed on the sixth or seventh day of seedling development coincides with a decrease in DNA and protein contents and apoptotic internucleosomal nuclear DNA fragmentation in the coleoptile. Incubation of seedlings in the presence of the antioxidant BHT (ionol) strongly affects their development but it does not influence the increase in DNA and protein contents for the initial four days of seedling life, and it slows down the subsequent age-dependent decrease in protein content and fully prevents the age-dependent decrease in DNA content in the coleoptile. A decrease in the O₂^·- amount induced by BHT distorts the seedling development. BHT retards seedling growth, presumably by suppression of cell elongation, and it increases the life span of the coleoptile. It seems that O₂^·- controls plant growth by cell elongation at the early stages of seedling development but later O₂^·- controls (induces) apoptotic DNA fragmentation and protein disintegration.
 
 

In recent years mechanism of internal initiation of translation in eukaryotic cells commands the attention of molecular biologists in increasing frequency. Ten years ago, experiments with picornaviruses demonstrated the ability of 40S ribosomal subunits to bind to nucleotide sequences localized far from the 5' ends of RNA molecules, and since then numerous viral and even cellular RNAs were shown to be capable of internal initiation of translation. In the present survey, data on the localization, structure, and functional load of these internal ribosome entry sites (IRES elements) of viral and cellular RNAs, as well as on proteins capable of strong and highly specific binding to IRES elements, are discussed. A conclusion is that a unified model of structure and functioning of viral and cellular IRES elements cannot be suggested.
 
 

A rapid and effective method for testing a repair enzyme, uracil-DNA-glycosylase, was proposed. As a substrate, a deoxyuridine-containing 5'-32P-labeled deoxyoligonucleotide covalently attached to a polystyrene support (Tenta Gel S-NH2) was used. The ammonia cleavage of the apyrimidine site formed in the enzymic reaction followed by the transition of the labeled oligonucleotide fragment from the solid phase into solution allowed the detection of the enzymic activity.
 
 

A new method of preparation of noncovalent complexes between poly(ethylene glycol) (PEG) and proteins (alpha-chymotrypsin (ChT), lysozyme, bovine serum albumine) under high pressure has been developed. The involvement of polymer in the complexes was proved using ⁽³⁾H-labeled PEG. The composition of the complexes (the number of polymer chains per one ChT molecule) depends on the molecular mass of PEG and decreases with the increase in molecular mass from 300 to 4000, whereas the portion of the protein (wt %) in complexes does not depend on the molecular mass of incorporated PEG and corresponds to approximately 70 wt %. The kinetic constants for enzymatic hydrolysis of N-benzoyl-L-tyrosine ethyl ester and azocasein catalyzed by the PEG-ChT complexes are identical with the corresponding values for the native ChT. According to the data obtained by the method of circular dichroism the enzyme in the complexes fully retains its secondary structure. The steric availability of PEG polymer chains in the complexes was evaluated by their complexation with alpha-cyclodextrin (CyD) or polymer derivatives of beta-CyD modified with PEG (PEG-beta-CyD). In contrast to free PEG, only part of PEG polymer chains (approximately 10%) interact with alpha-CyD. Thus, the complexation of PEG with ChT proceeds by means of multipoint interaction with surface groups of the protein globule located far from the active site and results in the sufficient decrease in the availability of polymer chains. The complexes between PEG chains in PEG-protein adducts and PEG-beta-CyD may be considered as a novel type of dendritic structures.
 
 

Heterocyclic bases and phosphate groups involved in the DNA-methyltransferase SsoII (M-SsoII) interaction were identified in the regulatory DNA region localized within the promoter region of the SsoII restriction-modification genes by footprinting with the use of formic acid, hydrazine, dimethyl sulfate, and N_ethyl-N-nitrosourea as modifying agents. It has been established that the enzyme interacts with three guanines, one adenine, two thymines, and three phosphate groups of each strand of the DNA duplex. These heterocyclic bases and phosphate groups are disposed symmetrically within the 15-mer inverted repeat of the regulatory DNA region. It has been demonstrated by footprinting with dimethyl sulfate that the C7 atoms of guanines interacting with the enzyme are exposed to the DNA major groove. Two theoretical models were built describing the contacts in a complex between M·SsoII and the regulatory DNA region.
 
 

A new purification procedure for the isolation of the "unlinking" enzyme, which hydrolyzes the phosphodiester bond between 5'-terminal uridylic acid of the encephalomyocarditis viral RNA and protein VPg has been developed. The enzyme (tyrosine-(5'P-->O)-uridylylpolynucleotide phosphodiesterase, Y-pUpN PDE) was purified from frozen mouse carcinoma Krebs II cells. The purification procedure included ammonium sulfate fractionation of the cell extract, pH fractionation by acidification of the protein solution to pH 4.0, cation-exchange chromatography on CM-52-cellulose, chromatofocusing, and size-exclusion HPLC on a TSK 2000 SW column. The enzyme was shown to exist as several forms characterized by different isoelectric points (ranging from 4.0 to 5.2) and molecular masses. The pH fractionation and ion-exchange chromatography on CM-cellulose influenced the pI and molecular mass values for each form (pI increased, whereas molecular mass decreased from 30 to 26 kD). The employment of these two stages removed (almost completely) an accompanying proteolytic activity, which co-purified with Y-pUpN PDE and digested free VPg. The molecular mass of 26 kD determined by HPLC for the native form coincided with the molecular mass of the major protein band determined by SDS-PAGE for the denatured form of the enzyme.
 
 

Modern data on the structure and function of transport/messenger (tm) RNA are reviewed. This stable RNA is involved in releasing ribosomes that are unable to complete protein synthesis on mRNA lacking the stop codon. The resulting abnormal proteins are rapidly degraded by specific proteases, which recognize a signal peptide encoded by the template region of tmRNA. The discovery of trans-translation has caused a particular interest in structural and functional studies of tmRNA.
 
 

Productive poliovirus infection of HeLa cells leads to the canonical cytopathic effect (CPE), whereas certain types of abortive infection result in apoptosis. To define the time course of commitment to the different types of poliovirus-induced death, inhibitors of viral replication (guanidine HCl) or translation (cycloheximide) were added at different times postinfection (p.i.). Early in the infection (during the first ~2 h p.i.), predominantly proapoptotic viral function was expressed, rendering the cells committed to apoptosis, which developed several hours after viral expression was arrested. In the middle of infection, concomitantly with the onset of fast generation of viral progeny, the implementation of the viral apoptotic program was abruptly interrupted. In particular, activation of an Asp-Glu-Val-Asp (DEVD)-specific caspase(s) occurring in the apoptosis-committed cells was prevented by the ongoing productive infection. Simultaneously, the cells retaining normal or nearly normal morphology became committed to CPE, which eventually developed regardless of whether or not further viral expression was allowed to proceed. The implementation of the poliovirus-induced apoptotic program was suppressed in HeLa cells overexpressing the Bcl-2 protein, indicating that the fate of poliovirus-infected cells depends on the balance of host and viral pro- and antiapoptotic factors.

Plant virus-encoded movement protein(s) (MP), and for many viruses the coat protein (CP), is required to mediate viral spread between plant cells via plasmodesmata (PD). Most probably, the genomic RNA of potexviruses moves through PD as assembled vifions and/or as ribonucleoprotein complexes containing the CP and 25-kDa MP. Here we report that encapsidated potato virus X (PVX) virion RNA, which is nontranslatable in a cell-free protein synthesizing system, can be converted into a fully translatable form after interaction of intact PVX particle with the PVX 25-kDa MP. The 25-kDa MP molecules bind selectively to only one extremity of the viral particle (that presumably contains the 5' ehd of the genomic RNA). The process of complex formation is ATP-independent; i.e., the ATPase activity of the 25-kDa MP is not involved'in the binding of the MP to PVX virion.
 
 

We determined nucleotide sequences of the VP1 and 2AB genes and portions of the 2C and 3D genes of two evolving poliovirus lineages: circulating wild viruses of T geotype and Sabin vaccine-derived isolates from an immunodeficient patient. Different regions of the viral RNA were found to evolve nonsynchronously, and the rate of evolution of the 2AB region in the vaccine-derived population was not constant throughout its history. Synonymous replacements occurred not completely randomly, suggesting the need for conservation of certain rare codons (possibly to control translation elongation) and the existence of unidentified constraints in the viral RNA structure. Nevertheless the major contribution to the evolution of the two lineages came from linear accumulation of synonymous substitutions. Therefore, in agreement with current theories of viral evolution, we suggest that the majority of the mutations in both lineages were fixed as a result of successive sampling, from the heterogeneous populations, of random portions containing predominantly neutral and possibly adverse mutations. As a result of such a mode of evolution, the virus fitness may be maintained at a more or less constant level or may decrease unless more-fit variants are stochastically generated. The proposed unifying model of natural poliovirus evolution has important implications for the epidemiology of poliomyelitis.
 
 

The coxsackie B3 virus oriR is an element of viral RNA thought to promote the assembly of a ribonucleoprotein complex involved in the initiation of genome replication. The mutual orientation of its two helical domains X and Y is determined by a kissing interaction between the loops of these domains. Here, a genetic approach was worked out to identify spatial orientation-dependent recognition signals in these helices. Spatial orientation changes (due to linear and rotational shifts) were introduced by appropriate insertions/deletions of a single base pair into one or both of the domains, and phenotypic consequences caused by these mutations were studied. The insertion of a base pair into domain Y caused a defect in viral reproduction that could be suppressed by a base-pair insertion into domain X. Similarly, a defect in viral replication caused by a base-pair deletion from domain X could be suppressed by a base-pair deletion from domain Y. Thus, certain areas of the two domains should cross-talk to one another in the sense that a change of space position of one of them required an adequate reply (change of space position) from the other. Phenotypic effects of the local rotation of one or more base pairs (and of some other mutations) in either domain X or domain Y suggested that the two most distal base pairs of these domains served as orientation-dependent recognizable signals. The results were also consistent with the notion that the recognition of the distal base pair of domain Y involved a mechanism similar to the intercalation of an amino acid residue.
 
 

Sonic properties of two Kazakh strains (K1 and K2) of tobacco mosaic virus (TMV) are described. K1 had been isolated by Dr M. Gordm in 1963, and K2 recently in our laboratory. Both strains were rather similar in host range and antigenic properties to the tomato strain of TMV (tomato mosaic virus, ToMV), but differed from the latter by inducing unusual symptoms on upper noninoculated leaves of infected tobacco plants. K1 was semi-defective and temperature-sensitive, and formed large amounts of long RNA-free helical protein rods in infected plants. K2 was found to be neither defective nor temperature-sensitive, and did not produce protein rods in infected cells. K1 and K2 coat protein gene sequencing data showed, as expected, that both proteins are similar in primary structure to ToMV coat protein: only three amino acid substitutions, relative to ToMV, were found in K1 and five in K2 coat protein. Two of these substitutions are unusual, namely, substitution of normally strictly conserved R92 by S (with concomitant Q99® R change) in K2 and substitution of K53 by E in K1.
 
 

A study was made of the coat protein (CP) of thermosensitive semidefective tobacco mosaic virus strain K1 (TMV-K1). In contrast to CP of other TMV strains, K1 CP showed high nonspecific aggregation and did not form normal two-layered cylindrical aggregates. In none of the conditions tested, K1 CP formed virions with cognate K1 RNA in vitro. The abnormal properties were attributed to substitution Lys53-Glu differentiating the K1 CP from those of other tobamoviruses. It is assumed that the high structural plasticity allows the tobamovirus virions to incorporate CP subunits even with unfavorable amino acid changes.
 
 

We studied subcellular distribution of green fluorescent protein (GFP)-tagged movement proteins encoded by the second and the third genes of poa semilatent hordeivirus (PSLV) triple gene block (TGB), 15K TGBp2 and 18K TGBp3. GFP-15K transiently expressed in Nicotiana benthamiana leaf epidermal cells was associated with the endomembrane system elements. GFP-18K appeared in the membrane bodies at cell periphery. Mutation analysis demonstrated that subcellular targeting of GFP-15K depended on the protein transmembrane segment(s), whereas the TGBp3 central hydrophilic region was responsible for targeting of GFP-18K. Coexpression of GFP-15K with the intact 18K protein induced drastic changes in the TGBp2 localization: GFP-15K appeared in the cell peripheral bodies similar to those in the cells expressing GFP-18K alone Coexpression experiments with mutant forms of both proteins argue against involvement of direct interaction between small TGB proteins in the TGBp3-assisted targeting of TGBp2 to the cell peripheral compartments. This conclusion was further confirrtied by similar effects on the PSLV 15K TGBp2 localization induced by TGBp3 proteins of PSLV and potato virus X, which have no detectable sequence similarity to each other.

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