Biomedical subjects

V M Stepanov

Publications and source records attributed to V M Stepanov.

At least 19 recordsLinked to original sources

PubMed2000

[Segmented condensation of peptides on a solid phase using subtilisin complexed with sodium dodecyl sulfate].

The subtilisin-sodium dodecyl sulfate complex was shown to catalyze the coupling of peptide segments on a solid phase in organic medium. By a two-stage enzymic condensation of peptide fragments on aminosilochrom (A) containing Met-Ala-Gly as a spacer, Dnp(or Boc)-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Met-Ala-Gly-A and Z-Ala-Ala-Glu(OMe)-Ala-Ala-Leu-Met-Ala-Gly-A were obtained. It was shown that the condensation products can be split off from the support using the Met residue cleavage by BrCN.

Peptides↗

PubMed1999

Synthesis of peptide aldehydes via enzymatic acylation of amino aldehyde derivatives.

Two ways for semi-enzymatic preparation of the peptide aldehydes are proposed: (1) enzymatic acylation of amino alcohols with acyl peptide esters and subsequent chemical oxidation of the resulting peptide alcohols with DMSO/acetic anhydride mixture or (2) enzymatic acylation of the preliminarily obtained by a chemical route amino aldehyde semicarbazones. Subtilisin 72, serine proteinase with a broad specificity, distributed over macroporous silica, was used as a catalyst in both cases. Due to the practical absence of water in the reaction mixtures the yields of the products in both enzymatic reactions were nearly quantitative. The second way seems to be more attractive because all chemical stages were carried out with amino acid derivatives, far less valuable compounds than peptide ones. A series of peptide aldehydes of general formula Z-Ala-Ala-Xaa-al (where Xaa-al = leucinal, phenylalaninal, alaninal, valinal) was obtained. The inhibition parameters for these compounds, in the hydrolysis reactions of corresponding chromogenic substrates for subtilisin and alpha-chymotrypsin, were determined.

Acylation↗

PubMed1999

Bacillus intermedius glutamyl endopeptidase. Molecular cloning and nucleotide sequence of the structural gene.

The glutamyl endopeptidase gene of Bacillus intermedius was cloned from a genomic library expressed in Bacillus subtilis and sequenced (EMBL accession number Y15136). The encoded preproenzyme contains 303 amino acid residues; the mature 23-kDa enzyme consists of 215 residues. The mature enzyme reveals 38% of identical residues when aligned with the glutamyl endopeptidase from Bacillus licheniformis, whereas only five invariant residues were found among all known glutamyl endopeptidases. The amino acid residues that form the catalytic triad (H47, D98, and S171) as well as H186 participating in the binding of the substrate carboxyl group were identified. It seems that the structural elements responsible for the function of glutamyl endopeptidases from various sources are highly variable.

Amino Acid Sequence↗

PubMed1999

A new subtilisin-like proteinase from roots of the dandelion Taraxacum officinale Webb S. L.

A serine proteinase from roots of Taraxacum officinale Webb S. L. was isolated by affinity chromatography and gel-filtration on Superose 6R using FPLC. The enzyme is a 67-kD glycoprotein containing 54% carbohydrate which we have named taraxalisin. The substrate specificity of taraxalisin toward synthetic peptides and oxidized insulin B-chain is comparable with that of cucumisin from Cucumis melo and the subtilisin-like serine proteinase macluralisin from Maclura pomifera. The proteinase is inactivated by DFP and PMSF. Taraxalisin exhibits maximal activity at pH 8.0. The pH range for stability of the enzyme is narrow--6.0-9.0. The temperature optimum for the subtilisin-like activity is 40 degrees C. The N-terminal sequence of taraxalisin has 40% of its residues identical to those of subtilisin Carlsberg. Thus, the serine proteinase from dandelion roots is a member of the subtilisin family, which is evidently widespread in the plant kingdom.

Amino Acid Sequence↗

PubMed1999

Two types of entomocidal crystals of Bacillus thuringiensis ssp. finitimus have the same set of unique delta-endotoxins.

The strain B-1166 differs from the other strains of Bacillus thuringiensis ssp. finitimus because it has two crystal types with different localization in the sporulating cell, i.e., inside and outside of exosporium membrane. Two dissociants of the strain were obtained containing only one of the crystal types. The initial strain produces at least three various delta-endotoxins (Fin2, Fin3, and Fin5) differing from all other known entomocidal proteins; Fin2 and Fin3 are similar to each other but differ from Fin5. Both crystal types contain the same endotoxins (Fin2, Fin3, and Fin5). In the B-1166 strain the site of crystal deposition is not determined by their protein composition.

Amino Acid Sequence↗

PubMed1999

Membrane proteins of Aedes aegypti larvae bind toxins Cry4B and Cry11A of Bacillus thuringiensis ssp. israelensis.

Proteins of molecular weight 65 and 62 kD and having affinity for toxins Cry4B and Cry11A produced by Bacillus thuringiensis ssp. israelensis have been isolated from brush border membranes of Aedes aegypti larvae using affinity chromatography. Using a ligand blotting technique, we show that the binding of these proteins to the biotinylated toxins is reversible and that the two toxins compete for binding to the two proteins. These proteins are likely to be Cry4B and Cry11A toxin receptors in gut epithelial cells of Aedes aegypti larvae.

Aedes↗

PubMed1999

[Formation of dipeptides--a side reaction in racemization of phenylalanine].

It was shown that acetylated dipeptides, Ac-D-Phe-D-Phe-OH, Ac-L-Phe-L-Phe-OH, Ac-D-Phe-L-Phe-OH, and Ac-L-Phe-D-Phe-OH, are formed during D-phenylalanine racemization. The overall content of these dipeptides in the reaction mixture ranged from 40 to 60% depending on the reaction conditions. We concluded that, like alpha-aminoisobutyric acid, phenylalanine is prone to polymerization under racemization conditions.

Chromatography, High Pressure Liquid↗

PubMed1998

Taraxalisin -- a serine proteinase from dandelion Taraxacum officinale Webb s.l.

Latex of dandelion roots contains a serine proteinase that hydrolyzes a chromogenic peptide substrate Glp-Ala-Ala-Leu-pNA optimally at pH 8.0. Maximal activity of the proteinase in the roots is attained in April, at the beginning of plant development after the winter period. The protease was isolated by ammonium sulfate precipitation of the root extract followed by affinity chromatography on a Sepharose-Ala-Ala-Leu-mrp and gel filtration on Superose 6R performed in FPLC regime. Pure serine proteinase named taraxalisin was inactivated by specific inhibitors of serine proteinases, diisopropylfluorophosphate (DFP) and phenylmethylsulfonylfluoride (PMSF). Its molecular mass is 67 kDa and pI 4.5. pH stability range is 6-9 in the presence of 2 mM Ca2+, temperature optimum is at 40 degrees C; Km=0.37+/-0.06 mM. The substrate specificity of taraxalisin towards synthetic peptides and insulin B-chain is comparable with that of two other subtilisin-like serine proteinases, cucumisin and macluralisin. The taraxalisin N-terminal sequence traced for 15 residues revealed 40% coinciding residues when aligned with that of subtilisin Carlsberg.

Amino Acid Sequence↗

PubMed1998

Expression of the carboxypeptidase T gene from Thermoactinomyces vulgaris in stable protoplast type L-forms of Proteus mirabilis.

The structural gene of the carboxypeptidase T (cpt) was successfully expressed in cell wall-less L-form cells of Proteus mirabilis. The DNA sequence encoding the PhoA leader peptide was fused with a truncated cpt gene encoding the mature enzyme. The modified gene in a pUC-based kanamycin resistance vector under the control of the lac promoter was transformed into L-form cells of P. mirabilis. They were able to produce the recombinant CpT both as a secretory and as a cell-bound insoluble form. The co-secretory processing of the PhoA leader peptide was quite efficient. The yield of the secreted CpT was not less than 20 mg l-1 and should be improvable.

Amino Acid Sequence↗

PubMed1998

Limited proteolysis of Bacillus thuringiensis CryIG and CryIVB delta-endotoxins leads to formation of active fragments that do not coincide with the structural domains.

Bacillus thuringiensis "true" toxins consist of three domains: the N-terminal, alpha-helical domain followed by two beta-structural domains. Their limited proteolysis does not proceed at the domain boundaries, but is directed to the loops within the domains. There are at least two patterns of the limited proteolysis of "true" toxins. The first pattern, observed for CryIA and CryIVD delta-endotoxins, results in the proteolysis of the loops connecting beta-strands of the second domain. The second pattern, detected for CryIG and CryIVB proteins, consists in the cleavage of the loop connecting the fifth and the sixth alpha-helices of the first domain. The choice between the routes depends on the size, sequence, and dynamics of the loop that define its accessibility to a proteinase. Bioassay of CryIG and CryIVB delta-endotoxin fragments indicates that only two alpha-helices, the sixth and the seventh within the first domain, followed by the two beta-structural domains are sufficient for the insecticidal activity.

Aedes↗

PubMed1998

A new PCR-based approach to a fast search of a wide spectrum of cry genes from Bacillus thuringiensis strains.

A pair of highly degenerated primers was adapted to carry out a single-step PCR-detection of any known and probably unknown cry genes of classes cry1, cry4 and cry9 encoding for 130 kDa protein delta-endotoxins in the natural Bacillus thuringiensis (BT) strains. The Southern hybridization of the product has demonstrated that essentially remote cry-genes like cry1Aa and cry9A (cryIG) could be represented in the single amplificate if they are simultaneously present in the genome of the analyzed strain. Four genes were detected by the proposed scheme in the BT ssp. galleriae 11-67. One of them, gene cry1Ga1 was originally found and cloned using the PCR-amplification product obtained from the genomic DNA of this strain as a probe. The new gene was completely identical to one cloned by B. Lambert (unpublished, EMBL accession number Z22510) and essentially related to cryIM (EMBL accession number Y09326), renamed according to the new nomenclature as cry1Ga2.

Bacillus thuringiensis↗

PubMed1998

[Isolation of trypsin PC from the Kamchatka crab Paralithodes camtschatica and its properties].

Trypsin PC from the hepatopancreas of the king crab Paralithodes camtschatica was isolated and purified to apparent homogeneity by ion-exchange chromatography on Aminosilochrom and DEAE-Sephadex and affinity chromatography on arginine-agarose. The yield of the enzyme was 37.7%, and the purification degree was 21. Trypsin PC has a molecular mass of 29 kDa and pI < 2.5. It hydrolysis N-benzoyl-L-arginine p-nitroanilide at the optimum pH of 7.5-8.0 and at the temperature optimum of 55 degrees C (K(m) = 0.05 mM). Trypsin PC retained its activity within the pH range of 5.8-9.0 in the presence of Ca2+. The enzyme was inhibited by the specific inhibitors of serine proteases diisopropyl fluorophoshate and phenylmethylsulfonyl fluoride, by the trypsin inhibitor N-tosyl-L-lysylchloromethylketone, and by the trypsin inhibitors from soybean and potato. Trypsin PC was found to hydrolyze amide bonds formed by carboxylic groups of lysine and arginine in peptide substrates. The N-terminal sequence of this enzyme is IVGGTEVTPG.

Amino Acid Sequence↗

PubMed1998

[Synthesis of new chromogenic substrates for aspartyl proteases].

A general method was developed for the synthesis of new chromogenic substrates of aspartyl proteases: Dnp-Ala-Xaa-Phe-Phe-Ala-Arg-NH2, where Xaa was Ala or Ser. The synthetic scheme involved both chemical and enzymic stages, the condensation of tripeptides in an organic medium by means of pepsin immobilized on Celite being among the latters. The influence of organic solvents, reaction time, and the composition and ionic strength of the buffers used in the reaction mixture and at the pepsin immobilization step on the efficacy of the pepsin-catalyzed synthesis was studied.

Alanine↗

PubMed1998

[Determination of activity of aspartic proteinases by cleavage of new chromogenic substrates].

Chromogenic hexapeptides Dnp-Ala-Ala/Ser-Phe-Phe-Ala-Arg-NH2 containing a Phe-Phe bond, which is sensitive to aspartic proteinases, were used as substrates for assaying the activity of pepsin, chymosin, and aspergillopepsin A. The assay was performed after the separation of hydrolyzates on SP-Sephadex by measuring at 360 nm the absorbance of the dinitrophenylpeptide lacking the cationic group, which was formed upon the cleavage of the substrate. The kinetic parameters of the hydrolysis of the substrates were evaluated. It is shown that replacing the Ala residue with Ser in the P2 position does not substantially change the kinetic parameters. The substrates were hydrolyzed by pepsin several times faster than by aspergillopepsin A or chymosin. The method is sensitive and enables the activity of aspartic proteinases to be determined easily.

Alanine↗

PubMed1998

[Synthesis of three- and tetrapeptides catalyzed by subtilisin suspensions in organic solvents].

The behavior of subtilisin 72 in some aprotic solvents (acetonitrile, dioxane, and tetrahydrofurane) was studied. The enzyme was shown to be partially soluble in tetrahydrofurane, but it is rendered profoundly inactive in this solution. In acetonitrile and dioxane, subtilisin formed dilute suspensions whose activities were measured after dilution with water. Under these conditions, subtilisin suspended in acetonitrile manifested an activity that was an order of magnitude higher than that of its dioxane suspension, and this activity continued for a long time. Z-Ala-Ala-Leu-pNA was synthesized from Z-Ala-Ala-OCH3 and Leu-pNA under the catalysis by dilute suspension of subtilisin in acetonitrile. p-Nitroanilides of tetrapeptides, Z-Ala-Ala-P1-P'1-pNA, where P1 and P'1 were either Leu or Phe, were similarly synthesized in acetonitrile under catalysis by dilute subtilisin suspension at [S]:[E] = 10(5):1. p-Nitroanilides of tripeptides, Z-Ala-Ala-Leu-pNA, Z-Ala-Ala-Phe-pNA, and Z-Ala-Ala-Phe-NH2, were also synthesized in the presence of a concentrated subtilisin suspension at [S]:[E] = 10(3):1. It was shown that the increase in enzyme concentration resulted in the double coupling of nucleophile, and Z-Ala-Ala-Leu-Leu-pNA, Z-Ala-Ala-Phe-Phe-pNA, and Z-Ala-Ala-Phe-Phe-NH2 were obtained with 13, 33, and 40% yields, respectively. Therefore, such reaction systems can be used for creating long hydrophobic peptides whose synthesis in water-organic mixtures is difficult due to the poor solubility of starting components in aqueous buffer solutions.

Acetonitriles↗

PubMed1998

Purification and characterization of P33 protein of Candida utilis homologous to bgl2p of Saccharomyces cerevisiae; comparative analysis of the role of these proteins in molecular organization of the yeast cell walls.

P33 protein was isolated from the cell walls of Candida utilis. Homology between P33 and Bgl2p proteins from the cell walls of Saccharomyces cerevisiae was shown. The important role of these proteins in molecular organization of yeast cell walls was demonstrated using trypsin proteolysis and the "gene disruption" method.

Candida↗

PubMed1997

Glutamyl endopeptidase of Bacillus intermedius, strain 3-19.

A homogeneous glutamyl endopeptidase splitting peptide bonds of glutamic, rarely of aspartic acid residues in peptides and proteins, was isolated from Bacillus intermedius 3-19 culture filtrate using chromatography on CM cellulose and Mono S. The enzyme molecular mass is equal to 29 kDa, pI 8.4. The protease is inhibited by diisopropylfluorophosphate. The enzyme, like other glutamyl endopeptidases, reveals two pH optima at pH 7.5 and 9.0 for casein and one at pH 8.0 for Z-Glu-pNA hydrolysis. A 6 mM K(m) is found for hydrolysis of the latter substrate. The enzyme activity optimum lies at 55 degrees C, and it is stable at pH 6.5-11.0. Its N-terminal sequence shows 56% coinciding residues when compared with that of Bacillus licheniformis glutamyl endopeptidase.

Amino Acid Sequence↗

PubMed1997

A novel delta-endotoxin gene cryIM from Bacillus thuringiensis ssp. wuhanensis.

A new cryI-related sequence designated cryIM was cloned using an immunoscreening technique from ssp. wuhanensis of Bacillus thuringiensis (BT), previously reported to produce multiple Cry proteins [Chestukhina et al. (1994) Can. J. Microbiol. 240, 1026-1034]. Analysis of the cryIM nucleotide sequence revealed an ORF, BTII-type promoter-like sequence, peculiar for such genes, a translation initiation element and a putative transcription terminator. Nevertheless, its product was not previously found in the crystals of the host strain [Chestukhina et al. (1994) Can. J. Microbiol. 240, 1026-1034] which shows its weak or absent natural expression. The amino acid sequence of 1151 residues encoded by the continuous reading frame of cryIM is not identical but is essentially similar to the other delta-endotoxins of the CryI class. An IS231-like sequence was found 400 bp downstream of the cryIM stop codon and a fragment of the cryIAb gene was located 3 kb upstream of its initiator codon in the same orientation. Artificial expression of the cloned gene in E. coli under the control of the lacZ promoter allowed us to obtain its hypothetical protein product.

Amino Acid Sequence↗