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V M Stepanov

Publications and source records attributed to V M Stepanov.

At least 109 records · Page 6Linked to original sources
PubMed1983

Purification and properties of serine protease from Halobacterium halobium.

Pure extracellular serine protease was isolated from the culture filtrate of Halobacterium halobium by bacitracin-Sepharose affinity chromatography. The enzyme activity was completely and irreversibly lost if the NaCl concentration fell below 2 M. The protease consists of one polypeptide chain with a molecular weight of 41,000. It is characteristically enriched in Asx and Glx content, whereas the level of basic amino acids in the enzyme molecule is unusually low. The protease shows a preference for leucine in the carboxylic side of the scissile bond of the substrate, cleaving the B-chain of oxidized bovine insulin only at the Leu15-Tyr16 bond and liberating p-nitroaniline from L-pyroglutamyl-L-alanyl-L-alanyl-L-leucine-p-nitroanilide.

Amino Acids↗
PubMed1983

[Subtilisin-like proteinase SSPB from Streptomyces spheroides, strain 35].

A serine proteinase possessing a fibrinolytic activity was isolated from a culture filtrate of Streptomyces spheroides, strain 35. A consecutive use of affinity chromatography on bacillichin-silochrome and bacitracin-sepharose and ion-exchange chromatography on anionie PAP and cationic KMT resulted in a homogeneous proteinase with 1060-fold purification and 19% yield. The enzyme has a molecular weight of 28000; its amino acid composition is Asp31, Ser28, Thr29, Glu9, Pro14, Gly35, Ala42, Val26, Ile14, Leu13, Met2, Tyr9, Phe4, Trp3, His6, Lys4, Arg10. The enzyme has a pI at pH greater than 10 and the activity optimum against Z-L-Ala-L-Ala-L-Leu-pNA at pH 10-11. The enzyme is stable within the pH range of 4-11 and in 6 M guanidinium chloride pH 8.0 in the presence of Ca2+. The enzyme is inhibited by diisopropylfluorophosphate and benzylsulfofluoride, specific inhibitors of serine proteinases as well as by potato proteinase inhibitor. The serine proteinase SSPB isolated from Str. spheroides, strain 35 can be related to subtilisin-like serine proteinase, especially to those of SGPD and SGPE of Str. griseus.

Amino Acids↗
PubMed1983

[Extracellular serine proteinase A of Streptomyces rutgersensis].

Extracellular serine proteinase of Streptomyces rutgersensis (proteinase A) has been isolated by affinity chromatography on bacitracin-Sepharose in the presence of Na2EDTA. The proteolytic activity of the enzyme is completely suppressed by specific inhibitors of serine proteinases, phenylmethylsulfonylfluoride and diisopropylfluorophosphate, but is insensitive to Na2EDTA and sodium p-hydroxymercuribenzoate. The enzyme activity towards hemoglobin is maximal at pH 9-10, the maximal stability is observed within the pH range of 6-10. The molecular mass as determined by SDS-pore gradient electrophoresis is equal to 20000 +/- 2000. Proteinase A is similar to the proteinases A and B of Streptomyces griseus in terms of i) amino acid composition--Asp17Thr22Ser23Clu7Pro6. X Gly30Ala13Cys4Val18Met1Ile5Leu9Tyr10Phe6Trp1His2Arg7, ii) amino terminal sequence--Leu-Ser-Gly-Gly-Asp-Ala-Ile-Tyr-Ser-Asn-Ser-Ser-Xaa-Xaa-Ser-Leu-, iii) specificity of insulin B-chain hydrolysis, and iiii) ability to exhibit proteolytic activity in 8 M urea or 6 M guanidinium chloride. The enzyme lyzes the cells of Candida utilis. Besides proteinase A, S. rutgersensis produces at least two other proteolytic enzymes.

Amino Acid Sequence↗
PubMed1983

Proteinase affinity chromatography on bacitracin-Sepharose.

Antibiotic-cyclopeptide bacitracin covalently bound to Sepharose proved to be an efficient general ligand for affinity chromatography of aspartyl, serine, and metalloproteinases from various sources. The yields of purified enzymes varied from 50 to 180%. New experimental data extend the application of bacitracin-Sepharose for affinity chromatography of cysteine proteinases--papain, bromelain, and ficin. Hence, bacitracin acts as a ligand which more or less efficiently binds proteinases that belong to all the main classes of these enzymes. Bacitracin, being a weak proteinase inhibitor of broad specificity, interacts with the substrate-binding sites of proteinases, which explains its efficiency as a ligand.

Affinity Labels↗
PubMed1982

[Specific features of intracellular serine proteinase of Bacillus amyloliquefaciens on native and denatured protein substrates].

The effects of intracellular serine proteinase from B. amyloliquefaciens and of extracellular subtilisin BPN' on native and denaturated protein substrates were compared. The substrate hydrolysis by the enzymes was determined by a method initiating possible participation of intracellular serine protinease in intracellular protein degradation. This approach consists in a prolonged treatment of the products obtained after proteolysis with carboxypeptidase A with a subsequent amino acid assay. It was found that intracellular serine proteinase is capable of degrading denaturated protein substrates e. g. reduced, carboxymethylated ribonuclease A and bovine serum albumin, with an efficiency comparable to that achieved by subtilisin hydrolysis. On the other hand, the intracellular enzyme attacks the native proteins much slower than substilisin.

Amino Acids↗
PubMed1982

[Extracellular serine proteinase from Bacillus licheniformis].

The serine proteinase from B. licheniformis was purified by affinity chromatography on the sorbent obtained by attachment of p-(omega-aminomethyl)-phenylboronic acid via an amino group to CH-Sepharose. The use of this sorbent specific to the serine proteinases active sites resulted in a 35-fold purification of the enzyme with an apparent activity yield of 288%. Such a high activity yield is due to a removal of the enzyme inhibitors. The N-terminal sequence of B. licheniformis extracellular serine proteinase traced for 35 amino acid residues coincides with that of subtilisin Carlberg, a serine proteinase presumed to be secreted by a B. subtilis strain. Since the amino acid composition as well as the functional properties of these two enzymes did not reveal any noticeable differences, it was assumed that both proteinases are very similar, if not identical. This conclusion leads to reconsideration of the existing concept on an extremely fast rate of subtilisin evolution. Three multiple forms of B. licheniformis extracellular serine proteinase were found to differ only in their net charges, presumably as a result of partial deamidation of Asn or Gln residues within their structure.

Amino Acid Sequence↗
PubMed1982

[Differences in primary structures of delta-endotoxins produced by various serotypes of Bacillus thuringiensis].

The primary structures of delta-endotoxins (crystal-forming proteins) produced by two serotypes of Bacillus thuringiensis--V (var. galleriae) and III (var. alesti) were compared. These proteins differ by the specificity of their action on Lepidoptera larvae as well as by their molecular weights. To evaluate the homology of primary structures the tryptic hydrolysates of both endotoxins were fractionated by ion-exchange and thin-layer cellulose chromatography with a subsequent amino acid determination in the hydrolysates of thus obtained the fraction. Only 10%, of the fractions gained from the tryptic hydrolysates of the two delta-endotoxins had similar amino acid composition. In control experiments on comparison of two hydrolysates of the same delta-endotoxins the percentage of fractions with similar amino acid composition was as high as 50%, which reflects the tendency of this approach to overestimate the extent of the differences between the two sequences. Hence the delta-endotoxins produced by two serotypes of Bacillus thuringiensis, being clearly homologous, reveal substantial differences in their amino acid sequences, which are dispersed along the whole polypeptide chain. These striking differences in the primary structures are indicative of an unusually high rate of their evolution, which may be of functional importance for B. thuringiensis serotypes adaptation to different ecological niches.

Amino Acid Sequence↗
PubMed1981

Affinity chromatography of proteolytic enzymes on silica-based biospecific sorbents.

New biospecific sorbents for affinity chromatography of proteolytic enzymes were prepared by the attachment of the cyclopeptide antibiotics bacitracin, bacilliquin or gramicidin S to aminosilochrom via a reaction with p-benzoquinone. The content of the cyclopeptide ligands within the sorbents varied from 2 to 46 mumol/g. The sorbents prepared by this reaction were successfully applied in the purification of the carboxylic proteinases produced by fungi, Russula decolorans (a basidiomycete) and Trichoderma lignorum, as well as crude pepsin. Serine proteinases from Thermoactinomyces vulgaris, Trichoderma koningii, Trichoderma lignorum and bacilli (subtilisins) were also submitted to chromatography on these materials. The yields of purified enzymes approached quantitative levels, sometimes being higher as a result of elimination of inhibitors. An important advantage of these sorbents is their stability against the enzymes degrading the carbohydrate matrixes of affinity sorbents synthesized on the basis of agarose, dextran or cellulose derivatives.

Animals↗
PubMed1981

[Carboxylic proteinases from the microscopic fungi Trichoderma viride and Trichoderma lignorum].

Using ion-exchange chromatography on aminosilochrome and biospecific chromatography on Bacitracin-Sepharose, the carboxylic proteinases have been isolated for the first time from the microscopic fungi of the Trichoderma genus -- Trichoderma viride and Trichoderma lignorum, commonly used to produce cellulases. The proteinases are stable within the pH range of 3 to 6; pI is 4,3 and 4,5, the pH optimum -- 2,3 and 2,8, respectively. The molecular weight of the enzymes is 32000, the amino acid composition of T. viride proteinase is Lys5His2Arg6Asx27Thr39Ser38Glx27Pro13..Gly41Ala28Cys2Val37Ile11Leu17Tyr13Phe11Trp3, that of T. lignorum is Lys9His4Arg6Asx36Thr26Ser46Glx25Pro14Gly35Ala23Cys2Val28Ile26Leu19Tyr12..Phe14Trp4. Both enzymes are completely inactivated by specific inhibitors of carboxylic proteinase, i. e. pepstatin, diazoacetyl-D,L-norvaline methylester and N-diazoacetyl-N'-2,4-dinitrophenylethylenediamine. The molecular and enzymatic properties of the proteinases under study are close to those of carboxylic proteinases of microscopic fungi and in a lesser degree to those of porcine pepsin.

Amino Acids↗
PubMed1981

[Modification of two tyrosine residues in aspergillopepsin A by p-nitrophenyldiazonium chloride].

p-Nitrophenyldiazonium chloride was found to modify the Tyr-75 and Tyr-189 residues in aspergillopepsin A. Incubation of the protein with a 45-fold molar excess of the reagent at pH 5,2 results in the attachment of 1,8 inhibitor residues, which leads to a 75% decrease of the enzyme activity towards haemoglobin. The pepsin inhibitor--pepstatin--protects one of the tyrosyl residues against modification. The chromatography on Sepharose-4B-Bacitracin showed that aspergillopepsin modified at pH 5.2, consists of molecules, which contain one and two incorporated inhibitor residues. The enzyme containing one inhibitor residue possesses 35% of activity of native protein. Apergillopepsin containing two inhibitor residues is practically inactive. It is concluded that at least one of the modified tyrosyl residues is closely related to the enzyme activity.

Amino Acid Sequence↗
PubMed1981

[Intercellular serine proteinases from spore-forming bacilli].

Some physico-chemical properties, immunological reactions, structural, functional and evolutionary features of intracellular serine proteinases from spore-forming bacilli were studied. These enzymes belong to an individual subfamily of serine proteinases and in terms of their structure and evolution are most closely related to secretory subtilisins. Intracellular serine proteinases are found in a wide variety of microorganisms--from the spore-forming bacilli to Escherichia coli and are characterized by a higher (as compared to secretory subtilisins) rate of evolution due to a more rigid selective control of the structure and function of intracellular enzymes. Active intracellular proteinases have a unique dimeric structure, which allows to exclude random proteolysis of native proteins in vivo. Secretory subtilisins and intracellular bacillary serine proteinases are coded by separate closely related structural genes, whose presence in the bacillary genome can be explained by duplication of the precursor gene. The existence of duplicated genes of serine proteinases provides sufficient evidence for the marked structural divergence and a high rate of evolution of secretory subtilisins.

Amino Acid Sequence↗
PubMed1981

[Isolation and comparative properties of serine proteinases of the microscopic fungi Trichoderma lignorum and Trichoderma koningii].

Using affinity chromatography on bacitracin-Sepharose combined with ion-exchange chromatography on aminosilochrome and isoelectrofocusing, individual serine proteases have been isolated for the first time from surface cultures of T. lignorum and T. koningii. The proteinases have pI values at 6.8 and 6.7 and pH optima of 10.5. Both proteinases are stable within the pH range of 4-11 and have molecular weight of 21 000. The amino acid composition of T. lignorum enzyme is Lys3His4Arg9Asx23Thr17Ser23Glx10Pro8Gly27Ala25Cys3Val13Met2Ile11Leu11Tyr7Phe6Tr p3, that of the T. koningii enzyme is Lys3His4 Arg9Asx23Thr16Ser26Glx10Pro9Gly29Ala26Cys3Val14Met2Ile9Leu11Tyr6Phe5Trp3. The enzymes are completely inhibited by phenylmethylsulfonylfluoride, diphenylcarbamoylchloride and trypsin inhibitors from beans, Actinomyces janthinus and potato tubers. The enzymatic and molecular properties of the enzymes are similar to those of subtilisins and previously described fungal serine proteinases, especially to those of proteinase K of the fungus Tritirachium album Limber.

Amino Acid Sequence↗
PubMed1980

alpha-Bromo-4-amino-3-nitroacetophenone, a new reagent for protein modification. Modification of the methionine-290 residue of porcine pepsin.

A new coloured reagent for protein modification, alpha-bromo-4-amino-3-nitroacetophenone (NH2BrNphAc), was synthesized. The reagent was found to alkylate specifically the methionine-290 residue of porcine pepsin below pH 3 at 37 degrees C, which lead to a 45% decrease of enzyme's activity towards haemoglobin. The effect of this reagent as well as that of other phenacyl bromides on the activity of pepsin appeared to be a result of steric hindrance caused by the attachment of bulky reagent residue to the edge of the cleft harbouring the enzyme active site. Only marginal reaction with the co-carboxy group of aspartic acid-315 was found under the above conditions. More pronounced esterification of carboxy groups (up to one residue per enzyme molecule) occurred when the pH was shifted to 5.2. The latter modification had no noticeable effect on enzyme activity, thus disproving a previously held assumption that pepsin inactivation by phenacyl bromide is due to the carboxy-group esterification. alpha-Bromo-4-amino-3-nitroacetophenone forms derivatives with characteristic u.v. spectra when it reacts with methionine, histidine, aspartic and glutamic acid residues, and may be recommended as a reagent for protein modification.

Acetophenones↗
PubMed1980

Crystal-forming proteins of Bacillus thuringiensis. The limited hydrolysis by endogeneous proteinases as a cause of their apparent multiplicity.

The crystals of the entomocidal protein of Bacillus thuringiensis are admixed with proteinases that in the course of their dissolution cause gradual degradation of the "genuine" crystal-forming protein components (i.e. the primary biosynthetic products) to products of lower molecular weight. This phenomenon might explain at least partially the contradictory data on the molecular parameters of the crystal-forming proteins. Preliminary inactivation of the proteinases adsorbed on the crystals allowed us to eliminate this source of the artefacts and to gain more reliable data on the protein composition of the crystals formed by various strains of B. thuringiensis. It has been shown that the crystals formed by all serotypes of B. thuringiensis, with the exception of the serotype V, contain only one protein with a mol. wt. of 145000, 135000 or 130000, depending on the strain. The majority of the strains that belong to the serotype V form crystals consisting of two proteins with mol. wts. of 135000 and 130000, but some of them also have a third component with a mol. wt. of 65000.

Bacillus thuringiensis↗