Search PubMed⌕ Search

Biomedical subjects

V M Stepanov

Publications and source records attributed to V M Stepanov.

At least 127 records · Page 7Linked to original sources
PubMed1980

[Carboxylic proteinase from the basidiomycete Russula decolorans Fr 0456].

A carboxylic proteinase has been isolated from a cultural filtrate of the basidiomycet Russula decolorans Fr 0456. A sequential chromatography on biospecific adsorbents containing polypeptide antibiotics gramicidin S and bacitracin used as ligands resulted in a 186-fold purification and 48% yield of the enzyme. The molecular weight of the enzyme is 35 000, pH-optimum of hemoglobin hydrolysis is 2,2, the isoelectric point lies at 4,9. The enzyme revealed a pronounced milk-clotting activity and is completely inactivated by the specific inhibitors of carboxylic proteinases--N-diazo-acetyl-N'-2,4-dinitrophenylethylenediamine and pepstatin. Its amino acid composition was also found to be similar to that of the carboxylic proteinases isolated from microscopic fungi. The data obtained suggest that the carboxylic proteinase from the basidiomycet Russula decolorans Fr 0456 is closely related to pepsin-like carboxylic proteinases, particularly to those produced by microscopic fungi.

Amino Acids↗
PubMed1980

[Metalloproteinase from Bacillus subtilis: - "intracellular" and extracellular enzymes].

"Intracellular" metalloproteinase was purified to homogeneity from Bacillus subtilis 103 crude cell extract, using affinity chromatography on bacitracin-Sepharose 4B. The degree of purification and the yield of the enzyme were about 260-fold and 3%, respectively. In its physico-chemical properties and the amino acid composition the enzyme is very similar, if not identical, to the extracellular metalloproteinase isolated from the culture filtrate of the same strain. Extracellular metalloproteinase-deficient mutant strain Bacillus subtilis SMY-512 does not produce the "intracellular" enzyme either. THe activity of "intracellular" metalloproteinase in the periplasmic space of the cells is about 70% of that in the cytoplasm, thus being indicative of a rather regular distribution of the enzyme throughout the cell compartment.

Amino Acids↗
PubMed1980

[Isolation of proteinase I from E. coli cells using affinty chromatography on bacitracin-Sepharose].

A serine proteinase (proteinase I) was isolated in a homogeneous state from E. coli K12 cells, using bacitracin-Sepharose 4B affinity chromatography. The enzyme effectively cleaved N alpha-acetyl-L-phenylalanine beta-naphthyl ester. The proteinase was inhibited by diisopropylphosphofluoridate and phenylmethanesulphonyl fluoride, but was resistant to EDTA and natural trypsin or subtilisin protein inhibitors. The enzyme did not cleave trypsin and subtilisin synthetic substrates, possessing a narrow substrate specificity. The amino acid composition of the enzyme was determined. The enzyme molecular weight was found to be about 20 000.

Amino Acids↗
PubMed1980

[Carboxylic proteinase from Trichoderma lignorum].

A carboxylic proteinase has been isolated from a commercial preparation of Trichoderma lignorum used as a source of cellulolytic enzymes. The purification procedure included precipitation by (NH4)2SO4 (65% saturation), gel-filtration through Acrylex P-10, affinity chromatography on gramicidin S bound to an inorganic matrix, gel-filtration through Acrylex P-10, affinity chromatography on bacitracin-Sepharose and separation on Ultrogel AcA 54 followed by gel-filtration through Sephadex G-50. A 400-fold purification of enzyme was achieved, the enzyme yield being 7,2%. The molecular weight of carboxylic proteinase as determined by gel-filtration is 33 000; its amino acid composition is found to be similar to that of carboxylic proteinases isolated from other fungal species. The enzyme is stable within the pH range of 3,0-6,0. The enzyme was fully inhibited by the specific inhibitors of carboxyliec proteinases-N-diazoacetyl-N'-2,4-dinitrophenylethylenediamine and pepstatin.

Amino Acids↗
PubMed1980

[A serine proteinase from Thermoactinomyces vulgaris, strain INMI-4a].

A serine proteinase was isolated from the cultural filtrate of the thermophylic actinomycet Thermoactinomycet vulgaris, strain INMI-4a. The purification procedure included affinity chromatography on bacitracin-Sepharose, ion-exchange separation on aminosilochrome, and gel-filtration on Sephadex G-25, resulting in a 194-fold purification and the 55% yield of the enzyme. The molecular weight of the enzyme as determined by polyacrylamide gel electrophoresis in the presence of Na-SDS as well as by gel-filtration on Sephadex G-75 is equal to 28 000; the amino acid composition is: Lys11, His4, Arg5, Asp33, Thr22, Ser24, Glu16, Pro16, Gly30, Ala38, Cys1-2, Val20, Met1, Ile14, Leu8, Tyr16, The4, Trp6-7. The isoelectric point lies at pH 8--9; the pH optimum for the peptide substrate hydrolysis is Z-L-Ala-L-Ala-L-Leu-pNA is at 8.2. The enzyme is stable at pH 7--9. The temperature optimum of the proteolytic activity lies at 55 degrees; however, the enzyme is stable to heating for 1 h at 37 degrees. The proteinase is completely inactivated by the serine proteinase specific inhibitors--phenylmethylsulphofluoride and the protein inhibitor IT-AjT from Actinomyces, as well as by p-chloromercuribenzoate. The enzyme shows lytic activity against the cells of E. coli, Micrococcus lysodeicticus and of the yeasts. The Thermoactinomyces vulgaris serine proteinase, being definitely different from the serine proteinases from Actinomyces griseus, also reveals specific differences when compared to bacterial serine proteinases, e. g. subtilisins. There are some indications to the enzyme relationship with the family of carboxypeptidase Y-like serine proteinases.

Amino Acids↗
PubMed1979

Intracellular serine proteinase of Bacillus subtilis strain Marburg 168. Comparison with the homologous enzyme from Bacillus subtilis strain A-50.

Intracellular serine proteinase was isolated from sporulating cells of Bacillus subtilis Marburg 168 by gramicidin S-Sepharose 4B affinity chromatography. The enzymological characteristics, the amino acid composition and the 19 residues of the N-terminal sequence of the enzyme are reported. The isolated proteinase was closely related to, but not completely identical with, the intracellular serine proteinase of B. subtilis A-50. The divergence between these two intracellular enzymes was less than that between the corresponding extracellular serine proteinases (subtilisins) of types Carlsberg and BPN', produced by these bacterial strains. This may be connected with the more strict selection constraints imposed in intracellular enzymes during evolution.

Amino Acid Sequence↗
PubMed1979

The study of Escherichia coli proteases. Intracellular serine protease of E. coli-an analogue of bacillus proteases.

Two serine proteases in extracts of Escherichia coli grown to stationary phase were purified to homogeneity using affinity chromatography on gramicidin S-Sepharose 4B. One enzyme was closely related to, if not identical with, the 'trypsin-like' protease II of E. coli. The other was capable of cleaving the subtilisin chromogenic substrate N-carbobenzoxy-L-alanyl-L-alanyl-L-leucine-p-nitroanilide and resembled the intracellular serine proteases of Bacillus spp. The amino acid composition of this E. coli protease was similar to that of the Bacillus licheniformis enzyme. These data indicate a relationship between proteolytic enzymes of evolutionary distant Gram-negative Enterobacteriaceae and Gram-positive spore-forming Bacillus.

Amino Acids↗
PubMed1979

[Effect of penicillin series antibiotics on the properties of Brucella].

Investigation of the properties of antibiotic-resistant variants obtained experimentally from various species of Brucella indicated that benzylpenicillin, ampicillin and ceporin induced significant changes in the main properties of Brucella, i.e. phagolysability, agglutinability, production of brucellacin and hydrogen sulfate, attitude to dyes. Only the nutrient requirements of Brucella did not change when it was grown on synthetic media which is of great importance for investigation of changed Brucella strains.

Agglutination↗
PubMed1979

[Subtilisin 72: a serine protease from Bac. subtilis strain 72 - an enzyme similar to subtilisin Carlsberg].

Subtilisin 72, a serine proteinase secreted by Bac. subtilis strain 72 was purified by covalent chromatography on Sepharose sorbent containing p-(omega-aminomethyl)phenylboronic acid as a ligand. The homogeneity of subtilisin 72 was confirmed by isoelectrofocusing in a thin layer of polyacrylamide gel (pl 8.6). The amino acid composition of this enzyme is different from that of other subtilisins, e. g. subtilisin Carlsberg. The N = terminal amino acid sequence of subtilisin 72 traced up to the 35th residue turned to be the same as that of subtilisin Carlsberg with the exception of the 21st (Tyr) and the 30th (Ile) residues. This very pronounced extent of homology shows that subtilisin 72 is very similar although not identical to subtilisin Carlsberg.

Amino Acid Sequence↗
PubMed1979

[Chromatography of carboxylic proteinases on sorbents containing the 2,4-dinitrophenyl group. Role of ionic interactions].

The chromatography of porcine pepsin on biospecific sorbents (Sepharose-4B-epsilon-DNP-aminocapronylhydrazide and Sepharose-4B-N-DNP-N'-acetylhexamethylenediamine) was studied. The sorbents in question differ from the previously used hydrophobic sorbent Sepharose-4B-DNP-hexamethylenediamine by the lack of strongly basic groups in the site of the ligand binding to the polymeric matrix. No qualitative differences in the pepsin chromatography on the three sorbents were observed. Presumably the decrease of the pepsin binding to the sorbents, containing the dinitrophenyl group, at pH values above the isoelectric point may be due to the effects of the salt on the binding site in the enzyme molecule rather than to the screening of the positive charges of the sorbent by chlorine ions. A commercial preparation of pepsin was purified 2-fold on the sorbent Sepharose-4B-epsilon-DNP-animocapronylhydrazide. The synthesis of sorbents is described.

Carboxypeptidases↗
PubMed1979

[Affinity chromatography of subtilisin on a sorbent with epsilon-aminocapronyl-alanyl-alanyl-D-leucylamide. Detection of a serine protease with unusual properties].

A biospecific sorbent obtained by attachment of epsilon-aminocapronyl-L-alanyl-L-alanyl-D-leucylamide to CNBr-activated Sepharose 4B has been used for affinity chromatography of various samples of subtilisin BPN', e.g. subtilisin A ("Serva"), Nagarse, A-50. Two active components were isolated from subtilisin A (Serva"), the major component corresponding to subtilisin BPN' and the minor component (SII) being a serine proteinase with low molecular weight (about 10000). The molecular weight and amino acid composition of SII as well as the kinetic parameters of its action on peptide substrates (p-nitroanilides of N-benzyloxycarbonyl-Gly-Gly-Leu, -Ala-Ala-Leu, -Gly-Gly-Phe, -Ala-Ala-Phe. The low molecular weight proteinase possesses a high affinity for the leucine residue in P1 position and alanine in P2 and P3 positions. The specificity of this proteinase differs from that of the main component.

Chromatography, Affinity↗
PubMed1979

[Protein composition of crystals (delta-endotoxin) of different serotypes of Bac. thuringiensis].

The crystals of entomopathogenic protein from Bac. thuringiensis contain admixtures of proteinases adhering to their surfaces. A newly developed technique of protease inactivation allowed to estimate the true protein composition of the crystals of various strains of Bac. thuringiensis. It was shown that the crystals of all strains (with the exception of V and VIII) are composed of only one protein with molecular weights of 145,000, 135,000 and 130,000, depending on the strain. The crystals of serotype VIII and the majority of the V serotype strains have two proteins with molecular weights of 135,000 and 130,000. A method for estimation of the protein composition of crystal without their preliminary isolation from a crystal--spore mixture is proposed.

Bacillus thuringiensis↗