[Glycine receptors in nervous tissue and their functional role].

The literature data on glycine metabolism in neural tissue, mitochondrial Gly-cleaving system, Gly-catching system in neural and glial cells are summarized. The peculiarities of localization and distribution of specific glycine receptors and binding-sites in nervous tissue of mammals are described. Four types of glycine-binding receptors are described: own specific glycine receptor (Gly-R), ionotropic receptor, which binds N-methyl-D-aspartate selectively (NMDA-R), and ionotropic receptors of g-aminobutyrate (GABA A -R, GABA С -R). The features of glycine effects in neuroglial cultures are discussed.

[Contribution of blue-green pigments to hemolytic activity of Pseudomonas aeruginosa cultural fluid].

AIM: To assess the contribution of blue-green pigments of Pseudomonas aeruginosa to hemolytic activity of its cultural fluid. MATERIALS AND METHODS. Eight hospital strains and reference strain ATCC 15442 were used. Growth dynamics of strains as well as features of accumulation of hemolytic and phospholipase activity were studied. Purified samples of pyoverdin and pyocyanin were extracted by gel-chromatography and chloroform extraction methods. Hemolytic and lecitinase activities of the samples as well as effect of active oxygen scavengers and chelating agents on these activities were studied. RESULTS: Dynamics of accumulation of hemolytic activity significantly differed from that of phospholipase activity when strains were grown in liquid medium. Chromatographic separation of the pigments from cultural fluid supernatants sharply reduced its hemolytic activity. Purified samples of pyoverdin and pyocyanin were capable to lyse erythrocytes and chicken egg lecitin. These characteristics of the pigments were inhibited by nitroblue tetrazolium and sensitive to chelating agents. Conclusion. Pyoverdin and pyocyanin of pathogenic strains of P. aeruginosa are capable to lyse erythrocytes and suspension of purified chicken egg lecitin, they contribute to total hemolytic activity of pathogenic strains of Pseudomonas, which is not determined only by phospholipase C produced by microorganism. Lytic activity of the pigments is blocked by nitroblue tetrazolium and susceptible to some chelating agents. Apparently, this activity is mediated by superoxide radical and determined by presence of metals with transient valence in pigments' molecules.

[Effects of biogenic phosphates on protease-induced protein cleavage and functioning of plasminogen activators].

We showed, using the method of lysis of fibrin plates and five substrate proteins in a thin layer of agar gel, that inorganic orthophosphate (0.001-0.06 M) enhances by 50-250% the activatory functions of streptokinase, urokinase, and tissue plasminogen activator and, in general, by 1.2-12.0 times enhances protein lysis by trypsin, alpha-chymotrypsin, subtilisin, papain, bacterial metalloprotease, and even pepsin at a concentration < 4 mM. At higher concentrations, phosphate sharply inhibited pepsin activity and inhibited by 40-50% gelatin lysis by papain and gelatin (at a peak concentration) and casein lysis by metalloprotease. Inorganic pyrophosphate ions at concentrations of 10(-8)-10(-1) M enhanced the cleavage of a number of proteins by serine proteases and, at concentrations of 10(-5) -10(-3) M, the activities of pepsin, plasminogen tissue activator, and streptokinase by 100 and 40%, respectively. The pyrophosphate concentrations of > 10(-3) and >10(-4) M inhibited pepsin- and metalloprotease-induced lysis of virtually all proteins. ATP increased casein lysis by serine proteases, metalloprotease, and pepsin by 20-60% at concentration of 10(-3) M and by 30-260% at 10(-2) M concentration. At concentrations of 10-2 M, it inhibited the cleavage of some proteins by trypsin, chymotrypsin, papain, and metalloprotease by 20-100%, and, at concentrations of 10(-3) M, lysis of albumin with pepsin and other proteins (except for fibrinogen) by metalloprotease. A GTP concentration of 10(-7)-10(-2) M increased protein degradation by serine proteases, papain, and gelatin lysis by pepsin by 20-90%, whereas albumin lysis was inhibited by 40-70%. The presence of 10(-6)-10(-5) M GTP led to a slightly increased degradation of hemoglobin and casein by bacterial metalloprotease, while 10(-3) M GTP induced a drop in the activity of the metalloprotease by 20-50%. ADP could enhance gelatin lysis by trypsin, casein lysis by pepsin and papain, and inhibited metalloprotease activity by 20-100% (at 10(-3) M). Peculiarities of the effects of AMP and GD(M)P on gelatin lysis were found.

[Effects of plasminogen and streptokinase on the vital functions of nervous tissue cells in culture].

In the protein-deficient media plasminogen stimulated the vital functions of cells and in concentrations 10(-7)-10(-10) M it protected cells of sympathetic ganglia, neocortex and continues cell lines under damaging actions of H2O2 (0.0001 M), NH4CI (0.01 M) and cooling. Streptokinase essentially influenced the mode of damaging effect of ATP(0.001 M). Even a short-term exposition (20 min) of PC12 cells with both proteins (each in the concentration 10(-9) M) led to sharp alterations in intracellular ATP- or Ca(2+)-activated proteolysis. In some cases plasminogen and streptokinase provided acceleration of cultured tissue maturation, improvement of cell adhesion, high survival rate, the increase in quantity and length of processes and their arborisation. Electronic microscopy established the character of structural rearrangements of nervous tissue cells (neurons, astrocytes, oligodendrocytes), reflecting the protective action of plasminogen and streptokinase. In the presence of plasminogen and especially streptokinase, the total number of cultured glioma C6 and neuroblastoma IMR-32 cells, the intracellular contents of protein, RNA and DNA increased several-fold. Addition of plasminogen promoted formation of processes by neuroblastoma cells, this suggests initiation of differentiation of cellular elements. In cultures of sensitive and sympathetic ganglia streptokinase increased proliferation of Schwann cells. These proteins did not cause transformation of PC12 enterochromaffine cells to neurons, though plasminogen facilitated it. Plasminogen addition to cell cultures did not increase fibrinolytic activity of the culture medium in the culture medium, and streptokinase did not lose its plasminogen-activating capacity.

[Effects of plasminogen, streptokinase and their equimolar complexes with pyruvate kinase on the human neuroblastoma IMR-32 cells].

The system of extracellular proteolysing, consists of plasminogen (PGn), its active protease (plasmin), PGn activation and PGn activators inhibitors, influences the nervous tissue functions, their growth, differentiation and proliferation in both, normal and pathological conditions. The purpose of the investigation was to study the effects of exogenous PGn, its activator streptokinase (SK), PK and their equimolar complex on the morpho-functional state neuroblastoma IMR-32 cells. PGn, SK, PK and their complexes stimulated cells proliferation during 1-3 days of incubation, shown by cell quantity increase. We also observed DNA, RNA and protein increase. The low lactate dehydrogenase efflux was evidence of that an addition of the proteins under investigation in the culture medium prevented the development of degenerative alterations connected with serum deprivation. The levels of extracellular PGn-activator activity, as measured by the biochemical fibrinolytic assay, increased over SK. This SK effect vanished on the 3rd day when SK formed complexes with PK. New original facts obtained testify the probability of initiation of neoplastic transformation and tumor growth potentiation.

Effects of streptokinase on the development of rat cerebral cortical cells in vitro.

The aim of the present work was to study the effects of streptokinase (SK) on the ultrastructure of the cellular elements of the cerebral cortex of neonatal rats in vitro. Three series of cultures were used: cultures maintained in DMEM enriched with 15% fetal calf serum (control 1), some transferred to minimal nutritive medium containing only 0.5% serum (control 2), and some supplemented with SK (2000 MU/ml) (experimental). Addition of SK to the nutritive medium prevented a decrease in the viability of cells in a mature (14 days) dissociated culture from the neocortex of rat pups aged 1-2 days induced by transfer of the cultures to medium lacking serum proteins. The action of SK on 7-day cultures enhanced the loss of cell viability. Electron microscopy studies of organotypic cultures showed that at this concentration, SK prevented the development of destructive changes of astrocytes, oligodendrocytes, and neurons induced in explants by serum protein deficiency. Neurons showed an abundance of mitochondria, some of which had few cristae. Signs of destruction affected only the nuclei of neurons. After exposure to SK for 48 h, oligodendrocytes were activated (they contained an abundance of myelin-like bodies), with degradation of most astrocytes (surviving examples showing nuclear hyperchromicity). Neurons were resistant to these effects.

[Synthesis of modified fragments of fibrinogen and their effect on the activity of proteolytic enzymes].

New analogues of the Gly-Pro-Arg and Arg-Gly-Asp fragments of fibrinogen were synthesized: Gly-Pro-Arg-Pro (I), Gly-Pro-Arg-Pro-Met-OMe (II), Gly-Pro-Arg-Pro-Phe (III), Gly-Pro-Arg-Pro-Asp (IV), Gly-Pro-Arg-Pro-Glu (V), and Arg-Asn-Trp-Asp (VI). Their effect on the activity of proteases of various types was studied with the method of lysis of fibrin plates. All the peptides were found to inhibit plasmin activity (by 60-85%) and the gamma-subunit of nerve growth factor (by 55-93%). Tetrapeptide (VI) proved to be an effective inhibitor of tissue activator of plasminogen and the gamma-subunit of nerve growth factor (by 96 and 93%, respectively). The peptides exerted practically no effect on the activity of urokinase and moderately inhibited the activity of streptokinase [(III), (IV), and (VI)], papain [(I), (II), (IV), and (VI)], subtilisin [(V) and (VI)], alpha-chymotrypsin [(III), (V), and VI)], and Bacillus subtilis metalloprotease (VI). They inhibit trypsin [except for (I) and (III)] when applied on fibrin plates at a concentration of 1 x 10(-2) M, while, at a concentration of 1 x 10(-3) M, (I) and (II) induced an increase in proteolytic activity by 35 and 47%, respectively.

Some unusual manifestations of proteolysis.

In the article the results of the long-term researches revealing the essence of the following three phenomena are generalized. 1. Participation of active oxygen species (especially, superoxide radical) in activation of zymogens--plasminogen, trypsinogen, chymotrypsinogen, pepsinogen and in realization of the catalytic (proteolytic) function of a number of proteinases. The essence of the hypothesis of oxygen-dependent reactions of proteolysis is stated. As shown by the example of mouse brain homogenate fractions, the plasminogen-activating ability of the fractions can also be realized via this way. From the positions of these views the experimental facts obtained about the influence of streptokinase and plasminogen on vital activity of nervous tissue cells are analysed. 2. Suppression of proteolytic reactions by ATP: plasminogen-activating ability of streptokinase, gamma- and betasubunits of the nerve growth factor, proteolytic activity of Arthrobothrys longa proteinases, destroyed cells of Corynebacterium diphtheriae PW-8. In some cases a significant effect was reached at concentration of ATP < or = 0.001 M. The effect depends on protein substrates used. 3. The increase of fibrinolytic activity of the mitochondrial fraction of the mouse brain and liver, proteolytic activity of human lymphoblasts of transplanted lines in the presence of inorganic orthophosphate. Judging by the results of inhibitory analysis, it is not caused by the resynthesis of ATP in the system and has an independent character--"phosphate effect" in proteolysis. 4. The results of our researches of formation of stable equimolar complexes of streptokinase or plasminogen with enzymes of carbohydrate-energetic metabolism are briefly analysed. The results of researches of functional properties of the molecules of diphtheria toxin, the nerve growth factor and it subunits are summarized. A number of fundamental and applied consequences of these phenomena are considered.

[The effect of streptokinase on the development of rat cerebral cortex cells in vitro].

The aim of this study was to determine the effect of streptokinase (SK) on the ultrastructure of cellular elements in the cerebral cortex of newborn rats in vitro. Three series of cell cultures grown on DMEM were used, including those grown on the medium enriched with 15% fetal calf serum (control 1), cultures transferred to the depleted medium containing only 0.5% of this serum (control 2), and the experimental cultures, to which SK (2000 IU/ml) was added. Addition of SK to the medium prevented a reduction of the viability of mature (14 days) dissociated neocortical cell culture from 1-2-day-old rats, induced by a transfer of the culture to a blood serum protein-deficient medium. In a 7-day culture SK potentiated the decrease in the cell viability. In organotypical cultures, with the use of electron microscopy, it was found that SK in concentration used prevented the development of destructive changes in astrocytes, oligodendrocytes, and neurons of explants, induced by a deficit in serum proteins. The neurons contained numerous mitochondria, some of which had only a few cristae. Signs of destruction were observed only in neuronal nuclei. After exposure to SK for 48 hours an activation of oligodendrocytes (containing numerous myelin bodies) was noted which was accompanied by astrocyte disintegration (with hyperchromatic nuclei in the remaining cells). The neurons were resistant to SK exposure.

Effect of active oxygen species scavengers on fibrinolytic activity of some proteinases.

Scavengers of different active oxygen species affect fibrin plate lysis, catalysed by various proteinases, only at relatively high concentrations (> 10(-2) M). Singlet oxygen scavengers change proteinase activity insignificantly except for strong inhibition of pepsin and papain by sodium azide, but pepsin-by histidine, and fibrinolytic urokinase activity-by all used O2 delta 1 scavengers. Of all used scavengers of OH-radical only ethanol caused significant changes in the proteinases under study, except for alpha-chymotrypsin. The most strong inhibitory effect on proteinase activity was demonstrated by scavengers of superoxide radical. Thus, nitrotetrazolium blue strongly inhibited the activity of plasmin, urokinase (fibrinolytic activity), papain and pepsin. Catalase changed proteinase activity insignificantly, though it leads to total inhibition of pepsin activity at final 4.5 x 10(-4) M concentration. These facts and our previous findings on generating of active oxygen species by proteinases give us grounds to suppose that minor active oxygen species, endogenous for the "proteinase-substrate" system, can participate in the catalytic function of some proteinases.

The state of tryptophan-containing sites of human, bovine and rabbit plasminogens with changing solution pHs.

The state of tryptophan-containing sites is proved to be stable by intrinsic tryptophan fluorescence with pH 5-8, 7-9, and 6-9 in human, rabbit and bovine plasminogen molecules, respectively. With pH < 5.0 tryptophan-containing sites of human zymogen (in contrast to rabbit and bovine ones) undergo conformational transitions. With the shift of solution pH from 9 to 12 tryptophan-containing sites of human and rabbit plasminogens are partially disorganized, while tryptophanyls become more available for solvent. Tryptophan-containing sites of bovine plasminogen molecules are less mobile in structure during changes of solution pH.

[Structural organization of the streptokinase molecule]. / Strukturnaia organizatsiia molekuly streptokinazy.

Conformational changes of the tryptophan-containing sites of streptokinase, its secondary and tertiary structures under the effect of NaCl, urea, heating, pH solution shift were analysed. Data on the reversibility of streptokinase's conformational changes, the flexibility of its structure, the presence of domains in the molecule were considered.

[Mechanism of the cytologic action of streptolysin O: effect of scavengers of active oxygen species and complexons on erythrocyte hemolysis]. / Mekhanizm tsitoliticheskogo deistviia streptolizina O: vliianie perekhvatchikov aktivnykh form kisloroda i kompleksonov na gemoliz éritrotsitov.

The hemolysis of rabbit erythrocytes, initiated by streptolysin O purified samples, is appreciably suppressed in the presence of the scavengers of singlet oxygen (sodium azide, histidine, tryptophan), .OH-radical (ethanol, mannitol), O.(2-)-radical (nitrotetrazolium blue, streptokinase), chelating agents (o-phenanthroline, sodium diethyldithiocarbamate) and some oxidoreductants such as K3FeCN6 and riboflavin as well. The streptolysin O hemolytic effect is supposed to be realized due to the participation of active oxygen species and this phenomenon depends upon the presence of the transition metals in the system.

Conformation ability test of human, rabbit and bovine plasminogens and their specific interaction with streptokinase.

Human, rabbit and bovine plasminogens, having different sensitivity to streptokinase-activating action, differ, according to spectrophotometric titration, tryptophan fluorescence and circular dichroism spectroscopy, in the state of tyrosine and tryptophan residues, and in secondary and tertiary structures. Human plasminogen-streptokinase equimolar complex formation (according to gel chromatography) is accompanied by a differential ultraviolet spectrum. Difference spectroscopy is a convenient and adequate means of studying the formation of the said complexes. Streptokinase-human plasminogen complex formation is not hindered by partial substitution of water (20%) with ethanol or dimethylsulphoxide or by addition of 0.001 M sodium dodecylsulphate. The complex is not formed in 6 M urea, in solution, at pH less than 2.0 or approximately 12.0-13.0, or with bovine plasminogen. Circular dichroism and tryptophan fluorescence spectral pattern changes during streptokinase-plasminogen complex formation enable us to conclude that streptokinase secondary and tertiary structures undergo certain rearrangements in the framework of the complex, while tryptophan-containing sites of the molecule are not drastically changed. The data obtained enable us to presuppose formation of streptokinase-rabbit plasminogen complexes which differ from human plasminogen complexes with streptokinase.

[Physico-chemical properties of the thrombolytic compound longolytin]. / Fiziko-khimicheskie svoistva tromboliticheskogo preparata longolitina.

A preparation exhibiting high fibrinolytic activity and ability to activate plasminogen was isolated from cultivation medium of Arthrobotrys longa. Homogeneous protein, obtained after gel filtration on Sephadex G-100, had molecular mass 28,600, pI-3.68-3.74, optimum activity at pH 6.0-9.0 and temperature optimum at 37 degrees. The enzyme proved to be serine proteinase as shown by analysis using inhibitors; it required thiol groups.

On the plasminogen-activating function of streptokinase.

1. Several hypotheses have been advanced to explain the activating function of streptokinase. The predominant hypothesis suggests a stable equimolar streptokinase-plasmin(ogen) complex, activating free plasminogen by an active centre, which is located in the plasmin(ogen) part of the complex. 2. This hypothesis cannot explain a number of phenomena and certain accumulated experimental data, for example: rabbit and bovine plasminogen activation by streptokinase, not forming stable complexes with these plasminogens; possible activation with pH less than or equal to 2, in the presence of urea, during modification of streptokinase tyrosine residues, i.e. when these two proteins cannot form a stable complex. 3. On the basis of acquired experimental data the following concept is suggested: the activating function of streptokinase is oxygen-dependent and is realised with the help of superoxide radical due to the O(2-.)-generating ability of plasminogen and the O(2-.)-converting ability of streptokinase.

[Chemical modification of the functional groups of streptokinase B in the water-soluble carbodiimide-nucleophile system]. / Khimicheskaia modifikatsiia funktsional'nykh grupp streptokinazy v sisteme vodorastvorimyi karbodiimid--nukleofil.

Specific activity of streptokinase was decreased after incubation in the system containing I-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC)--ethylene diamine. Under these conditions about 8 free amino groups and 38 free carboxylic groups were modified in the streptokinase molecule. The protein conformation and the state of tryptophane residues were altered. The streptokinase modified by means of EDC-ethylene diamine treatment lost its ability to form a stable complex with human plasminogen.

[Modification of functional groups of the streptokinase molecule during photooxidation]. / Modifikatsiia funktsional'nykh grupp molekuly streptokinazy pri fotookislenii.

Photochemical oxidation with methylene blue as photosensitizer results in the destruction of one histidine residue in the streptokinase molecule. This process is characterized by the rate constant corresponding to the modification of free L-histidine and results in partial inactivation of the protein. The rate of protein photo oxidation and photoinactivation is pH-dependent. As can be judged from the results of CD spectroscopy and gel chromatography, in weakly acidic (but not in weakly alkaline) media the reaction results in conformation changes of the streptokinase globule which affect the state of the protein tryptophanyl residue. It was found that the imidazole group destroyed during the photooxidation reaction is not essential either for the specific activity of streptokinase or for the formation of is stable complex with human plasminogen. The specificity of modification of the streptokinase histidine residue during the photooxidation reaction is discussed.

[Study of the role of histidine residues in streptokinase molecule using modification with diethylpyrocarbonate]. / Issledovanie roli ostatkov gistidina v molekule streptokinazy s pomoshch'iu modifikatsii diétilpirokarbonatom.

The interaction of streptokinase with diethylpyrocarbonate resulting in partial inactivation of the protein was studied. Eight histidine residues are blocked per streptokinase molecule by this reagent. Ethoxyformylation of streptokinase histidyls is characterized by a rate constant corresponding to modification of free L-histidine. No reactivation of streptokinase was achieved by treatment of the modified protein with hydroxylamine. The CD spectroscopy data suggest that the residues modified by diethylpyrocarbonate are of no consequence for the stabilization of the protein secondary structure. The specificity of modification of streptokinase histidine residues by diethylpyrocarbonate is discussed. Based on the gel chromatography data, it was assumed that partial inactivation of streptokinase depends on the formation of protein oligomers with a decreased activatory function.

[Dynamics of proteins in a culture broth growing beta-hemolytic streptococci group C]. / Dinamika belkov kul'tural'noi zhidkosti pri roste beta-gemoliticheskikh streptokokkov gruppy C.

The dynamic study of the protein spectrum of culture fluid during the growth of beta-hemolytic streptococcal strain H46A has been carried out by the methods of electrophoresis and isoelectrofocusing in polyacrylamide gel. Changes in the protein spectrum have phasic character and, on the whole, reflect the state of the microbial population, the presence of fractions corresponding to streptokinase and streptolysins being detected at all phases of growth. The electrophoretic mobility of streptokinase perceptibly changes at the end of the logarithmic phase; as shown by electrofocusing, at all stages of the population growth the heterogeneity of streptokinase is observed.