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V M Stepanov

Publications and source records attributed to V M Stepanov.

At least 91 records · Page 5Linked to original sources
PubMed1990

[Thiol-dependent serine proteinase from Streptomyces thermovulgaris].

The cultural filtrates of S. thermovulgaris contain a proteinase which is active towards the chromogenic subtilisin substrate, Z-Ala-Ala-Leu-pNa, and azocasein. Pure enzyme preparations were obtained by affinity chromatography on bacitracin-Sepharose with subsequent rechromatography on the same adsorbent. The proteinase was completely inactivated by PMSF and DFP, the specific inhibitors for serine proteinase, by thiol reagents (HgCl2, PCMB) and by the protein inhibitor from S. jantinus. The pH activity optimum for the enzyme is 7.8-8.2, temperature optimum is 55 degrees C. The enzyme is stable at pH 6-9, has a pI of 5.0 and a molecular mass of 32 kDa. When tested against the peptide substrate, the enzyme shows a specificity characteristic for subtilisins. The N-terminal sequence of the enzyme, Tyr-Thr-Pro-Asn-Asp-Pro-Tyr-Phe-Ser-Ser-Arg-Gln-Tyr-Gly, shows a 100% homology with that of terminase, a thiol-dependent serine proteinase. On the basis of the above considerations the enzyme may be related to the subfamily of thiol-dependent serine proteinases.

Amino Acid Sequence↗
PubMed1990

[Blood groups of great gerbils (Rhombomys opimus Licht.) as an indicator of population resistance to infections].

Three genetic systems of blood groups of great gerbils were studied to reveal that different resistance patterns exist in rodents toward Yersinia pestis and Salmonella typhimurium. Extrapolation of the results obtained on relations of co-members of parasite systems contributes to understanding of some points of immunobiological mechanisms of dynamics of expizootic procedures. A hypothesis is proposed claiming that isoantigenic blood structure of the host is responsible for formation of population resistance to infection.

Animals↗
PubMed1989

[Enzyme activity of induced auxotrophic mutants of El Tor Vibrio cholerae].

Vibrio cholerae induced auxotrophic variants nonfermenting several carbohydrates simultaneously have been obtained for the first time. Eltor vibrio clones have been selected, referred to Heuberg's groups V and VI by their enzymic activities. The findings of the present study evidence that, when isolating Eltor vibrio strains negative towards differential carbohydrates, in case of a positive agglutination test, the incubation period should be prolonged to 3 days. The principal differential diagnostic test for this species, when cholera is bacteriologically diagnosed, should be a positive volumic agglutination test with species- and variant-specific sera, irrespective of Heuberg's group.

Genetics, Microbial↗
PubMed1989

[Cytolytic activity of Legionella pneumophila].

The properties of cytolysin and metalloproteinase purified by different methods have been studied. The physico-chemical properties of these proteins, including their molecular weight, immunodiffusion patterns, the degree of inhibition by EDTA and diethyl pyrocarbonate, amino acid composition, cytolytic and proteolytic activity, have proved to be similar. We have come to the conclusion that cytolysin and metalloproteinase have similar composition and metalloproteinase activity determines the cytolytic and necrotic activity of the above-mentioned cytolysin.

Amino Acids↗
PubMed1988

[Primary structure of intracellular serine proteinase from Bacillus amyloliquefaciens. I. Isolation of the enzyme and amino acid sequence of peptides of tryptic hydrolysate].

Method of isolation of intracellular serine protease was modified. Gramicidin S-sepharose CL-4B with a higher content of the ligand, synthesized through a modified procedure, was used as an affinity sorbent which simplified the purification and led to the pure enzyme with high specific activity and 90% yield. Trypsin hydrolyzate of the protease was separated by ion-exchange chromatography on a sulphocationite resin followed by paper chromatography and paper electrophoresis to yield twenty-five individual peptides. Their complete or partial sequences, corresponding in total to 146 amino acid residues, were determined by the manual Edman procedure.

Amino Acid Sequence↗
PubMed1988

[New fluorescent substrates for metalloendopeptidases with internal quenching of fluorescence].

p-Nitroanilides of antranyloyltripeptides of the general structure Abz-Ala-Ala-P'1-pNA (P'1 = Phe, Leu, Ile, Val) containing intramolecularly quenched fluorescent groups (Abz is a fluorogenic group and pNA is a quencher of fluorescence) were prepared by combination of chemical and enzymatic methods. Thermolysin and metalloproteinases from Legionella pneumophila and Thermoactinomyces species were shown to hydrolyse Ala-P'1 bond of the peptides with simultaneous 4-7 fold increase in fluorescence. Kinetic parameters for enzymatic hydrolysis of the substrates were determined. Metalloendopeptidases can be assayed in the presence of serine proteinases (of the subtilisin type) using Abz-Ala-Ala-Ile-pNA or Abz-Ala-Ala-Val-pNA.

Actinomyces↗
PubMed1987

[Synthesis of p-nitroanilides of acylated peptides catalyzed by thermolysin].

Thermolysin-catalysed synthesis of p-nitroanilides of acylpeptides of general formula Z-A1-A2-pNA (A1 = Thr, Ala, Val, Leu; A2 = Leu, Phe) and stepwise synthesis of p-nitroanilides of acyltetrapeptides of general formula Z-A1-A2-A3-A4-pNA (A1, A2 = Gly,Ala; A3, A4 = Ala, Leu, Phe) from Z-A1-A2-OH and A3-pNA and then from Z-A1-A2-A3-OH and A4-pNA have been carried out; pNA group was eliminated enzymatically. Increase in solubility of the product in the reaction mixture diminishes its yield. Minimal amount of thermolysin providing a substantial yield of reaction product depends on structure of both amino and carboxylic components. In many cases the molar ratio of the enzyme and starting substances could be decreased to 1:10(6) as compared with the generally used ration 1:10(3)-1:10(4).

Amino Acids↗
PubMed1987

[Extracellular metalloproteinase from Legionella pneumophila].

Using ion-exchange chromatography on QAE-Sephadex A-50, affinity chromatography on DNP-hexamethylenediamine-Sepharose and gramicidin S-Sepharose and gel filtration, a metalloproteinase was isolated from the cultural fluid of L. pneumophila (strain Philadelphia-1) grown for 20 hours. The enzyme was purified 1606-fold with a 31% yield. The enzyme has a Mr of 38,000, pI approximately 4.0 and optimum of proteolytic activity at pH 6.0-7.0, 55 degrees C. The proteinase is the most stable within the pH range of 6.0-9.0. The enzyme contains one atom of zinc per molecule. The amino acid composition of metalloproteinase is close to that of thermolysin and is characterized by a high methionine content--17 residues out of 348. In the B-chain of oxidized bovine insulin the enzyme hydrolyzes the bonds precedent to the amino groups of leucine, phenylalanine and tyrosine. The enzyme is inhibited by chelating agents--Na2-EDTA and o-phenanthroline as well as by diethylpyrocarbonate. The serine and thiol proteinase inhibitors do not influence the enzyme activity. Under the given conditions of cultivation metalloproteinase is the major endopeptidase produced by L. pneumophila. Thus, the proteolytic system of Legionelles is characterized by the combination of metalloproteinase and the earlier described phenylalanine aminopeptidase.

Amino Acids↗
PubMed1986

Proteinases of Legionella: phenylalanineaminopeptidase of L. pneumophila.

Phenylalanineaminopeptidase was isolated and purified from the culture filtrate of Legionella pneumophila by affinity chromatography on O-tert-butyl-L-threonyl-L-phenylalanyl-L-prolylglycyl-aminosilo chrom and by gel-filtration; a 401-fold purification with a yield of 18% was achieved. The enzyme was a metalloenzyme with a molecular weight of 35000 and a pI of 5.8. It was stable at pH 7-9 and had an activity optimum in the range of pH 8-9.5 with L-phenylalanine p-nitroanilide as substrate. Enzyme activity was highest towards the latter compound, substantially lower towards L-leucine p-nitroanilide and only marginal towards other p-nitroanilides. Besides phenylalanineaminopeptidase, a metalloproteinase and a serine proteinase were also detected in L. pneumophila culture filtrate.

Amino Acids↗
PubMed1986

[Modification of swine pepsin and pepsinogen by p-nitrophenyldiazonium chloride].

It was found that at pH 5.2 and 40-fold excess of p-nitrophenyldiazonium chloride the inhibitor incorporation into the porcine pepsin molecule involves 1.9 residues, one residue being bound to tyrosine 189. Besides, tyrosines 44, 113, 154 and 174 enter the reaction. Modified pepsin retains 25% of the native enzyme activity. In the pepsinogen molecule the degree of tyrosine 189 modification diminishes 5 times; of 1.5 inhibitor molecules incorporated into the protein 0.78 residues are bound to tyrosine 113. The potential proteolytic activity of modified pepsinogen towards haemoglobin cleavage makes up to 60% of the original one. It is concluded that the activation peptide in the pepsinogen molecule masks the substrate binding site bearing tyrosine 189, thus preventing its modification with p-nitrophenyldiazonium chloride. The activation peptide in the pepsinogen molecule is presumably located in the vicinity of the wide loop bend carrying tyrosine residue 113, which may be the reason for the decreased pKa value of this residue and of its increased reactivity in the azocoupling reaction.

Amino Acids↗
PubMed1986

[Modelling of rifampicin pharmacokinetics in experimental animals administered the drug intravenously and internally].

Pharmacokinetics of rifampicin on its single intravenous and oral administration to rats in doses of 25 and 50 mg/kg and on its intravenous administration to dogs in doses of 8 and 25 mg/kg was studied. When the antibiotic was administered intravenously to the animals, its pharmacokinetics was nonlinear. The linearity distortion in the rats was lower than in the dogs. However, the pharmacokinetic data relevant to the antibiotic administration in the above doses were satisfactorily described by the biexponential equation. The absolute extent of rifampicin systemic absorption following oral administration to the rats was 60 to 85 per cent. Tissue availability of the antibiotic on its intravenous administration was lower than that on its oral administration.

Absorption↗
PubMed1984

L-Pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide--a chromogenic substrate for thiol proteinase assay.

L-Pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA)--a convenient chromogenic substrate for assay of thiol proteinases papain, ficin, and bromelain--was prepared by enzymatic synthesis with chymotrypsin as a catalyst. The thiol proteinases hydrolyze PFLNA with the liberation of p-nitroaniline, estimated spectrophotometrically by its absorbance at 410 nm. The phenylalanine residue in the P2 position of PFLNA meets the specificity demands of thiol proteinases. The following values of Km were found for PFLNA hydrolysis: by papain, 0.34 mM; by ficin, 0.43 mM; by bromelain, 0.30 mM. This substrate was successfully applied to monitor thiol proteinase affinity chromatography on bacitracin-Sepharose, which resulted in a 2- to 4-fold purification from commercial preparations.

Bacitracin↗
PubMed1984

[Pharmacokinetics of dihydrostreptomycin, after intravenous administration as a liposomal preparation, in the blood serum and tissues of intact mice and those with generalized tuberculosis].

Distribution of 3H-dihydrostreptomycin ( DHS ) in the blood serum and organs of intact mice and mice with generalized tuberculosis was studied. The antibiotic was administered in the form of a liposomal preparation and solution. It was shown that the distribution pattern of DHS administered in the form of a liposomal preparation corresponded to that of liposomes, which were the drug carriers. The serum levels of DHS injected intravenously in the form of a liposomal drug in a dose of 1 mg per mouse in the intact animals were 19 and 17 times higher 1 and 3 hours after injection, respectively as compared to those provided by the solution. In animals with generalized tuberculosis, the respective figures were 5 and 1.9 times. The levels of DHS in the liver provided by the liposomes were 4-6 times higher at all the time intervals in both the groups of animals. The antibiotic levels in the spleen of intact and infected animals were 2.5-3 and 8-12 times higher, respectively. In the lungs of intact animals, the antibiotic levels were 2 times higher 1 hour after administration. In infected animals, the levels of the antibiotic in the lung tissue were 3 times higher in 1 and 3 hours. The DHS levels in the kidneys of infected and intact animals were similar after drug administration in the form of a solution and liposomal preparation. The extremely high difference in the DHS levels in the spleen tissues of the mice with generalized tuberculosis after drug administration in the form of a solution and liposomal preparation may be of a significant importance for sanation of the organ lymphoid system affected with tuberculosis.

Animals↗
PubMed1984

[Carboxypeptidase T--intracellular carboxypeptidase of Thermoactinomycetes--a distant analog of animal carboxypeptidase].

Carboxypeptidase T, an extracellular carboxypeptidase from Thermoactinomyces sp. was isolated and purified by affinity chromatography on bacitracin adsorbents. The enzyme homogeneity was established by SDS electrophoresis (Mr = 38 000) and isoelectrofocusing in PAAG (pI 5.3). Carboxypeptidase T reveals a mixed specificity in comparison with pancreatic carboxypeptidases A and B and cleaves with nearly the same efficiency the peptide bonds formed by the C-terminal residues of basic and neutral hydrophobic amino acids. The enzyme is insensitive to serine and thiol proteinase inhibitors but is completely inhibited by EDTA and o-phenanthroline. The maximal enzyme activity is observed at pH 7-8. With an increase of temperature from 20 to 70 degrees C the enzyme activity is enhanced approximately 10-fold. In the presence of 1 mM Ca2+ the enzyme thermostability is also increased. In terms of some properties, e.g. substrate specificity carboxypeptidase T is similar to metallocarboxypeptidase secreted by Streptomyces griseus. The N-terminal sequence of carboxypeptidase T: Asp-Phe-Pro-Ser-Tyr-Asp-Ser-Gly- Tyr-His-Asn-Tyr-Asn-Glu-Met-Val-Asn-Lys-Ile-Asn-Thr-Val-Ala-Ser-Asn-Tyr- Pro-Asn - Ile-Val-Lys-Thr-Phe-Ser-Ile-Gly-Lys-Val-Tyr-Glu-Gly-Xaa-Gly-Leu- coincides by 21% with that of pancreatic carboxypeptidases A and B. Thus, it may be concluded that these enzymes originate from a common precursor.

Amino Acid Sequence↗
PubMed1984

[Multiple forms of horse pepsin].

Using ion-exchange and affinity chromatography and isoelectrofocusing, eight forms of pepsin with pI 1.6, 1.8, 2.1, 2.3, 2.6, 2.8, 3.2 and 3.6, were isolated from horse gastric juice. The molecular weights, amino acid composition, N-terminal sequence and functional activity of these multiple forms were determined. Partial primary structure of tryptic peptides of pepsin with pI 2.3 was investigated. The analyzed partial sequences of the forms with pI 1.8, 2.1, 2.3, and 2.6 have identical structures which differ from the amino acid sequence of pepsin with pI 3.2 by four substituents. In terms of their functional activity, horse pepsins differ only insignificantly. Presumably, the pepsins under study (at least the forms with pI 1.8, 2.1, 2.3, 2.6 and 3.2) arose comparatively recently as a result of duplication of the common precursor gene and exist at an early stage of structural and functional divergence. As far as their primary structure and functional properties are concerned, these pepsins are more related to pepsin A than to other isoenzymes of gastric aspartyl proteinases of mammalia, e. g., gastricsin or chymosin.

Amino Acid Sequence↗