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V M Stepanov

Publications and source records attributed to V M Stepanov.

At least 145 records · Page 8Linked to original sources
PubMed1979

[Proteinases in growth and spore formation of Bacillus thuringiensis].

Some serine proteases and leucine aminopeptidases were detected inside and outside the cells during the analysis of three crystalline and two acrystalline strains of Bac. thuringiensis var. galleriae. The data obtained on the protease formation during growth and sporulation and the level of their activity are indicative of intracellular proteases involvement in spore- and crystal formation. The enzymes isolated from the culture medium do not probably take part in these processes. The intracellular enzymes may account for the different crystal protein composition of various strains due to limited proteolysis of crystal proteins in the course of biosynthesis.

Bacillus thuringiensis↗
PubMed1978

On the appearance of Bacillus subtilis intracellular serine protease in the cell membrane and culture medium. Comparison of the enzyme and other Bacillus subtilis serine proteases.

While about 80% of the cell-bound intracellular serine protease of Bacillus subtilis A-50 have been recovered in the soluble fraction upon disruption of cells, the rest of the enzyme was found to be associated with the membrane fraction. Soluble cytoplasmic intracellular serine protease, as well as membrane-bound serine protease liberated by non-ionic detergent treatment, have been isolated in a pure state and shown to be identical. The same protease might also be found extracellularly, due presumably to cell lysis or altered membrane permeability. Intracellular serine protease of Bacillus subtilis A-50 was clearly related to Bacillus subtilis serine proteases W1 and bacillopeptidase F described as extracellular enzymes.

Amino Acids↗
PubMed1978

Phenylboronic acid as a ligand for biospecific chromatography of serine proteinases.

Via attachment of p-(omega-aminoethyl)phenylboronic acid to CH-Sepharose in the presence of water-soluble carbodiimide, a new sorbent for the biospecific chromatography of serine proteinases was obtained. The sorbent was shown to be suitable for the purification of subtilisn, alpha-chymotrypsin and trupsin. It is assumed that the serine hydroxyl group at the active site of the enzyme forms, with the boronic acid moiety of the ligand, a structure that imitates transition enzyme--substrage complex. The presence of glycerol selectively improves the binding of serine proteinases, presumably because of stabilization of the tetrahedral state of the boron atom. Direct isolation of subtilisin from a Bacillus subtilis cultural filtrate on phenylboronic acid-containing sorbent gives a virtually homogenous enzyme (42-fold purification) in a nearly-quantitative yield.

Bacillus subtilis↗
PubMed1978

Intracellular serine protease of Bacillus subtilis: sequence homology with extracellular subtilisins.

Intracellular serine protease was isolated from stationary-grown Bacillus subtilis A-50 cells and purified to homogeneity. The molecular weight of the enzyme is 31,000 +/- 1,000, with an isoelectric point of 4.3. Its amino acid composition is characteristically enriched in glutamic acid content, differing from that of extra-cellular subtilisins. The enzyme is completely inhibited with phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid. Intracellular protease possesses negligible activity towards bovine serum albumin and hemoglobin, but has 5- to 20-fold higher specific activity against p-nitroanilides of benzyloxycarbonyl tripeptides than subtilisin BPN'. Esterolytic activity of the enzyme is also higher than that of subtilisin BPN'. The enzyme is sequence homologous with secretory subtilisins throughout 50 determined NH2-terminal residues, indicating the presence of duplicated structural genes for serine proteases in the B. subtilis genome. The occurrence of two homologous genes in the cell might accelerate the evolution of serine protease not only by the loosening of selective constrainst, but also by creation of sequence variants by means of intragenic recombination. Three molecular forms of intracellular protease were found, two of them with NH2-terminal glutamic acid and one minor form, three residues longer, with asparagine as NH2 terminus. These data indicate the possible presence of an enzyme precursor proteolytically modified during cell growth.

Amino Acid Sequence↗
PubMed1978

[Induction of auxotrophy in the brucellosis microbe under the effect of N-methyl-N'-nitro-N-nitrosoguannidine].

Mutagenic activity of N-methyl-N'-nitro-N-nitrosoguanidine with respect to Brucella melitensis was studied. In the course of this study the method of obtaining auxotrophic brucella mutants was elaborated. Numerous assortiment of auxotrophic mutants of different types was obtained. In a number of cases the acquisition of auxotrophy was accompanied by profound changes of the strain, such as the changes in morphology of colonies, in the biochemical activity, in the capacity of agglutination and in the virulence.

Brucella↗
PubMed1978

[Characteristics of the polysaccharide-containing somatic antigens isolated from the K-1 strain of the plague microbe and its antibiotic-resistant variants].

Immunochemical analysis of 2 polysaccharide-containing structures of the lypopolysaccharide of the plague causative agent (main somatic antigen and lipopolysaccharide) isolated from K-1 strain and a number of its antibiotic resistant mutants was carried out. It was shown that development of resistance to streptomycin alone or its combination with monomycin did not cause detectable changes in the monosaccharide composition and serological properties of the cultures tested. More significant changes associated with development of complex resistance, i.e. K-1 (Strr leads to Penr leads to Tetr) were accompanied by a decrease in the content of hexozamine and serological activity of the main somatic antigen determining the O-specificity of the lipopolysaccharide. Defective changes in the monosaccharide composition and serological properties of both the main somatic antigen and the polysaccharide were observed in the yellow variant of the streptomycin resistant mutant K-1.

Anti-Bacterial Agents↗
PubMed1977

[Isolation and properties of leucine aminopeptidase from Aspergillus oryzae].

Homogenious leucine aminopeptidase is purified from "oryzine"--mixture of enzymes produced by surface culture of Asperigillus oryzae using treatment with activated characoal, followed by DEAE-cellulose and hydroxylapatite chromatographies, Biogel P-100 gel-filtration and polyacrylamide-gel electrophoresis. The enzyme has pH optimum 9.0 and the molecular weight 37500 as estimated by gil-filtration through Sephadex G-100 (superfine) and SDS-polyacrylamide gel electrophoresis. Leucine aminopeptidase from Asp. oryzae has a broad substrate specificity, therefore, cleaving with the highest rate the peptides carrying N-terminal leucine. The enzyme is completely inhibited with EDTA and beta-mercaptoethanol, and it is a metalloenzyme.

Aspergillus↗
PubMed1977

[Proteins of Bacillus thuringiensis delta-endotoxin crystals].

Pure crystals (at least 99% purification) of sigma-endotoxin were isolated from Bac. thuringiensis var. galleriae. The complete dissolution of crystals might be achieved by the increase of pH up to 12 and higher or by a combined action of S = S-reducing and denaturing agents. Electrophoresis of the solubilized crystal proteins in 5% polyacrylamide gels containing 0,1% sodium dodecyl sulfate and 8 M urea reveals two major bands corresponding to molecular weights of 120000--140000 (65%) and 65000 (8-10%), and some minor components whose molecular weights varied from 65000 to 340000. Urea (3--8 M) causes to partial dissolution of the crystals; the component with molecular weight of 65000 is mainly found in the solution (component A). In dithioerythritol extracts at pH 9 the major component of the crystal is the protein with molecular weight 120000--140000 (component B). The crystals, alkali-soluble components and proteins isolated from crystals by selective extraction (3--8 M urea or 0.01 M dithioerythrytol, pH 9) were found toxic for the larvae of Galleria mellonella.

Animals↗
PubMed1977

[Limited proteolysis of bovine pepsin].

A limited proteolysis of bovine pepsin (EC 3.4.4.1) was carried out. A proteolysis-resistant C-terminal protein fragment containing about 170 amino acid residues was isolated and its N-terminal sequence was established, using Edman's automatic method. It was assumed that the fragment of bovine pepsin isolated, similar to the previosly obtained porcine pepsin fragment, is an independent constituent of the protein molecule.

Amino Acid Sequence↗
PubMed1977

[Effects of mutations in Bacillus subtilis genome decreasing the protease activity on the formation of subtilisin molecular forms].

Multiple molecular forms of subtilisin--extracellular serine protease produced by the wild strain Bac. subtilis A-50 and its mutant strains with the protease activity decreased two-fold and more were studied. Six molecular forms of subtilisin were found on the whole when 33 mutant strains have been investigated under the experimental conditions. It is essential that both the wild and each of mutant strains under study produced not more than three out of these six forms. Three molecular forms of subtilisin from the mutant strains are similar to those found in the wild strain A-50, and have the molecular weight, of 27 000-30 000. Three other forms of subtilisin were revealed only in the mutant strains, and had the molecular weight of about 20 000. Apparently there is only one structural gene for subtilisin in Bac. subtilis genome. The appearence of multiple molecular forms of subtilisin may be due to the post-translational modifications (limited proteolysis) of the initial type of enzyme, i.e. pre-subtilisin. Probably, that certain mulations not affecting the structural gene can significantly change the expression of such gene by varying of the degree of product modifications.

Bacillus subtilis↗
PubMed1977

[Isolation of purified aspergillopepsin A].

Two methods of isolation of pure aspergillopepsin A have been developed. The first method is based on sequential chromatography of a crude preparation on gramicidin C-sepharose 4B and ECTEOLA-cellulose (35% yield, 100-fold purification). The second method consists in sequential chromatography of evaporated extract of Asp. awamori surface culture on Acrylex P-10, aminosilochrome and ECTEOLA-cellulose (40% yield, 430-fold purification). The methods discussed may be used to isolate pure aspergillopepsin A preparations in order to establish the primary structure of the enzyme.

Aspergillus↗
PubMed1977

[Experimental study of the mutagenic activity of gentamicin sulfate in relation to the causative agent of plague].

Gentamicin sulfate was studied with an aim of obtaining mutants of the plague microbe with a changed character of requirements in the growth factors. The antibiotic provided a high yield of auxotrophic mutants and induced streptomycin resistance in some cells. Mutants with single dependence on arginine, leucine, histidine and uracyl were most often. Comparison of the data obtained with gentamicin and those obtained with other chemical mutagens showed that gentamicin by its mutagenic activity was only slightly inferior than the preparations of the group of supermutagens.

Gentamicins↗
PubMed1977

Intracellular proteinase of Bacillus subtilis.

An intracellular serine proteinase was isolated from Bacillus subtilis, strain A-50. The molecular weight of the enzyme is 30000 +/- 1000, its amino acid composition is enriched in glutamic acid residues, the isoelectric point is 4.3, the N-terminal sequence Glu-Leu-Pro-Glu-Gly-Ile-Gln-Val-Ile-Lys-Ala-Pro-Glu-Leu-Xxx-Ala-Gln-Gly-Phe-Lys Gly-Ser-Asx-Ile-Lys-Ile-Ala-Val-Leu-Asx. The enzyme is structuraly homologous with secretory subtilisins.

Amino Acids↗
PubMed1976

[Structure of a pepsin fragment containing the functionally important aspartic acid residue].

The structure of B-5 fragment obtained under degradation of pig pepsin by cyanogen bromide is determined. B-5 fragment is a central part of pepsin molecule and it contains 46 amino acids, including a functionally important aspartic residue of the active site of the enzyme. Amino acid sequence of B-5 fragment was established by means of sequenator analysis and of studying the structure of peptides isolated from B-5 fragments.

Animals↗