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Biomedical subjects

V K Kibirev

Publications and source records attributed to V K Kibirev.

At least 19 recordsLinked to original sources
PubMed2000

Solid-state conjugation of proteins with hydrophobic compounds in non-denaturing conditions. I. Acylation of proteins by dansyl proline using a polymeric N-hydroxysuccinimide ester.

A method of conjugating protein molecules under native conditions with water-insoluble hydrophobic compounds is developed. It permits very water-insoluble acids to be gently coupled to the primary amines on proteins or other biopolymers. For this purpose we synthesized a polymer (co-polymer of N-hydroxymaleimide and N-vinylpyrrolidone cross-linked by benzidine) which swells equally well in water and in organic solvents. Hydrophobic substances are first activated by esterification to this polymer in organic solvent and then conjugated to protein by acylation in aqueous medium at pH 8.0-8.5. Thus, the contact of native protein with organic co-solvent may be completely avoided. The application of this approach is demonstrated by labeling trypsinogen and plasminogen with dansyl proline.

Acylation↗
PubMed1998

[New donor-acceptor pairs for fluorogenic substrates with intramolecular fluorescence energy transfer for thrombin and trypsin].

New substrates for thrombin and trypsin are described: a fluorogenic substrate Abz-Pro-Arg-Gly-Nph (I), whose action is based on intramolecular fluorescence energy transfer, and H-D-Trp-Pro-Arg-pNA (II), which can be used both as a chromogenic substrate and as a substrate with the intramolecular fluorescence energy transfer. In substrate (I), a 4-nitrophenylhydrazide group was first used as an acceptor of excitation energy of the 2-aminobenzoyl group. The substrate is poorly hydrolyzed by thrombin (kcat/K(m) = 1.4 x 10(3) M-1 s-1) and is efficiently cleaved by trypsin (kcat/K(m) = 3.15 x 10(6) M-1 s-1). The hydrolysis of (II) can be monitored both spectrophotometrically, by absorbance at 405 nm, and from the increase in fluorescence at 340 nm. In the efficiency of hydrolysis with thrombin (kcat/K(m) = 3.0 x 10(6) M-1 s-1), compound (II) is comparable with the known chromogenic substrates for this enzyme. The proposed donor-acceptor pairs are promising in designing substrates with the intramolecular fluorescence energy transfer for a variety of proteolytic enzymes.

Energy Transfer↗
PubMed1996

Synthesis and study on a novel inhibitor of thrombin containing no side chain at the P1 position of the polypeptide chain.

The presented work allows one to speculate that the hydrophobic contacts of the residues located at the P2 and P3 positions with the corresponding subsites of thrombin (S2 and S3) allow the synthesis of compounds that can react with thrombin specifically and probably may not interact with other trypsin-like serine proteases. Substitution of proline by m-Abz-residue may result in the development of novel substrates and inhibitors for thrombin.

Animals↗
PubMed1988

[Chromogenic and fluorogenic peptide substrates of proteolytic enzymes].

The review describes approaches to designing chromogenic and fluorogenic substrates for proteolytic enzymes, mainly for assay of serine proteinases. Principles of substrate polypeptide chain construction and some methods for detection of chromogenic and fluorogenic products of their hydrolysis are considered. The use of these substrates for the study of blood clotting enzymes and for clinical diagnostics is briefly treated. Methodology of chemical synthesis of principal chromogenic and fluorogenic substrates is also discussed.

Animals↗
PubMed1983

[Analysis of the relationship between the reactivity of synthetic substrates and thrombin inhibitors and their structure].

Kinetics of thrombin- and trypsin-catalyzed hydrolysis of diphenylacetyl-L-arginine esters was studied at pH 8.5 and 25 degrees C, and the antithrombin activity of in vitro synthesized compounds was examined. The anticlotting activity of arylsulphonyl-L-arginine methyl esters appeared to be higher than that of the derivatives of diphenyl arginine. Relations were found connecting polar (delta) and steric (Es) characteristics of substituent (R) in R-C6H4-SO2-Arg-OCH3 esters with their antithrombin activity in vitro or with efficiency of their thrombin-catalyzed hydrolysis. This gives supplementary possibilities for synthesis of new substrates and more potent thrombin inhibitors.

Arginine↗
PubMed1983

[Structural bases of thrombin specificity. Role of secondary interactions].

The action of thrombin on the esters, Tos-P2-Arg-OCH3 and Tos-P3-P2-Arg-OCH3, where P2 and P3 are the residues of L-, D- or N-methylamino acids, has been studied. The values of kcat and Km(app) under steady-state conditions at pH 8.5 have been determined. The results obtained and literature data suggest that one of the causes of the low catalytic potency of thrombin is a decrease in the hydrophobicity of the environment of Asp-102 located at the active site of the enzyme. Therefore thrombin can cleave only the peptides which hydrophobically shield Asp-102. The key role in this process may be played by the side chains of the amino acids at P2 and P9, thus suggesting the presence of a beta-turn in the P7-P4 region of the polypeptide substrates of thrombin.

Hydrogen-Ion Concentration↗
PubMed1982

[Separation of a bradykinin-releasing enzyme from the proteolytic complex of Levantine viper venom].

Two serine hydrolases have been separated from the proteolytic complex of the venom of Levantine viper, Vipera lebetina turanica. The enzyme with mol. weight of 50,000 +/- 5,000, pH-optimum of 8.5 and isoelectric point in the range of 5.6--6.6 had proteolytic activity against casein and hydrolyzed benzoyl-arginine p-nitroanilide. The other enzyme with mol. weight of 37,000 +/- 2,000, pH optimum of 9 and isoelectric point in the range of 4.1--4.5 had no effect on benzoylarginine p-nitroanilide, casein or hemoglobin, but possessed a bradykinin-releasing activity. Both enzymes were stereoselective against L-arginine, hydrolyzing tosyl-L-arginine methyl ester without having any effect on D-arginine ester. The interaction of the enzymes with a number of N(alpha)-arylsulfonylarginine methyl esters has been studied. The influence of the substitute X in the arylsulfonyl part of the substrates upon their hydrolysis by the bradykinin-releasing enzyme has been described by the Hammett equation of rho omicron = 1.14 +/- 0.33 (r = 0.974).

Animals↗
PubMed1981

[Effect of temperature on the hydrolysis of some synthetic substrates by thrombin and trypsin].

The effect o temperature on the thrombin- and trypsin-catalyzed hydrolysis of N(alpha)-arylsulfonyl-L-arginine methyl esters (I) and N(alpha)-methoxyphenylsulfonyl-L-valyl-L-arginine methyl ester (II) at pH 8.5 was studied. The non-linearity of the Arrhenius plots of the rate constants for thrombin can be due to changes in the rate-limiting step of the enzymatic reaction. The activation parameters delta G not equal to, delta H not equal to, delta S not equal to were determined. It was shown for both thrombin and trypsin that unfavourable alterations of the entropy of activation decreased with a transition from the substrates (I) to compound II. It was assumed that the low efficiency of hydrolysis of synthetic substrate by thrombin is probably caused by the lack of conformation changes in the enzyme active site, which is probably necessary for a specific catalysis by thrombin.

Enzyme Activation↗
PubMed1979

[Comparative study of hydrolysis of methyl esters of N-arylsulfonyl-valyl-arginine by thrombin and trypsin].

The esterase action of thrombin and trypsin on N-arylsulfonyl-valyl-arginine methyl esters was studied. The values of Km and kcat under steady-state conditions at pH 8,5 were determined. It was shown that the nature of the arylsulfonyl group does not affect the kinetic parameters of the reactions under study. The Michaelis constants of the thrombin-catalyzed reactions appeared to be one order of magnitude lower than the Km values of the corresponding TAME analogs.

Arylsulfonates↗
PubMed1978

[Determination of the kinetic parameters of individual stages of M(alpha)-arylsulfonyl-L-arginine ester hydrolysis by thrombin and trypsin].

The N(alpha)-arylsulfonyl-L-arginine ethyl- and propyl esters were synthesized and the kinetics of their hydrolysis by thrombin was studied. The values of kcat and Km were shown to depend on the structure of the leaving group and to decrease in the line: OCH3 greater than OC2H5 greater than OC3H7. Using methanol as an additional nucleophile, the kinetic parameters - k2, k3 and Ks - were measured for both thrombin- and trypsin-catalysed reactions. A similarity of two enzymes at the stage of Michaelis complex formation was revealed: the Ks values for both enzymes were practically identical (18.10(-5)M). The differences between thrombin and trypsin were observed at the stages of chemical conversion of substrates and were especially well-pronounced at the stage of acylation. It was shown that the k2 values for thrombin were lower than that for trypsin and the k2/k3 ratio of TAME hydrolysis by trypsin was equal to 21, while that for thrombin was 4.5. This finding is indicative of an essential role of the acylation step in thrombin-catalysed hydrolysis of the esters under study.

Arginine↗
PubMed1977

[Dependence of thrombin- and trypsin-catalyzed hydrolysis of N-alpha-arylsulfonyl-L-arginine methyl esters on the structure of acylamide part of substrates].

For comparative studies on the esterase activities of thrombin and trypsin N(alpha)-arylsulfonyl-L-arginine methyl esters were synthetised containing in aromatic ring substituents of different polar nature, size and hydrophobicity. The kinetics of their hydrolysis by thrombin and trypsin were measured. Values of Km and kcat in steady-state conditions were determined. It was shown, that thrombin-catalysed hydrolysis was more sensitive than that of trypsin to the nature of substituents of arylsulfonyl group and determined by their polar and steric effects. A line correlation between specificity constants (kcat/Km) and sigma and Es of substituents were demonstrated. The difference in reactivity of compounds under investigation is suggested to depend on alterations of stability of hydrogen bond between arylsulfonylamide nitrogen atom of substrate and the active center of the enzyme due to changes in the acidity of the arylsulfonylamide group affected by substituent of the benzene ring.

Arginine↗
PubMedDate not supplied

[Natural and synthetic inhibitors of thrombin. I. Natural inhibitors].

Structures and properties of main physiological (heparin, antithrombin III, heparin cofactor II) and nonphysiological (hirudin, thrombin-binding aptamers, cyclotheonamides) natural thrombin inhibitors and its fragments and synthetic analogs (hirugen, hirulogs, hirunorms, pentasaccharides, macrocyclic alpha-keto amides) were reviewed. The molecular mechanisms of interaction of these compounds with thrombin and their anticoagulant activity at preclinical and clinical trials are discussed. The examined of natural thrombin inhibitors and their synthetic fragments and analogs are perspective for prophylaxis and treatment of different thrombo-embolic diseases.

Antithrombin III↗
PubMedDate not supplied

[Natural and synthetic inhibitors of thrombin. II. Synthetic low-molecular-weight inhibitors].

The up-to-date problems, concerning the structure and properties of two types of inhibitors are reviewed. It is particularly considered properties of low-molecular weight thrombin inhibitors that have electrophilic groups capable to react with Ser-195 of thrombin (peptidyl-chloromethyl ketones, aldehydes, ketomethylene derivatives and derivatives of boric and phosphoric acids) and the competitive reversible thrombin inhibitors. The review focuses on methods of modification of the structure in the natural inhibitors and design of new peptidomimetics. The prospects for prophylaxis and treatment of diverse thromboembolic diseases are discussed.

Antithrombins↗
PubMedDate not supplied

[Synthesis and study of biological activity of stereoisomeric dipeptides containing tyrosine and arginine residues].

Series of methyl esters of stereoisomeric dipeptides of the sequences Tyr-Arg and Arg-Tyr has been synthesized by classic methods of the peptide chemistry. The study of their reactivity towards thrombin and trypsin has shown that the kinetic parameters of enzyme-catalyzed hydrolyses of stereoisomeric compounds differ in values essentially. Testing the synthetic peptides on analgic effect, on inhibition of the reaction fibrinogen with thrombin or on influence upon the process of fibrin-monomer polymerization has shown that these biological effects depend on peptide structure and on configuration of amino acid residues forming the peptides.

Antithrombins↗
PubMedDate not supplied

[Synthesis and antithrombin activity of phosphoalanine-containing peptides].

Boc/Tos-L-Phe-L-Arg-Xaa tripeptides (where Xaa = L-Ala-OBut, L-Ala, or DL-AlaP (OC2H5)2) were synthesized by conventional methods of peptide synthesis in solution. Special features of their interaction with thrombin and trypsin were studied. Unlike trypsin, thrombin did not catalyze the hydrolysis of the L-Arg-L-AlaP-(OC2H5)2 bond. The Tos-L-Phe-L-Arg-DL-AlaP(OC2H5)2 peptide was the most active inhibitor of thrombin among all the compounds studied. The relationship between the structure and inhibitory action of the synthesized peptides is discussed. A part of this study was reported in the Augustusburg Conference of Advanced Science: Nucleic Acids--Targets and Tools, September 17-19, 2000, Germany.

Alanine↗
PubMedDate not supplied

[Kinetics of trypsin-catalyzed hydrolysis of some arginine-containing peptide methyl esters].

The kinetics of trypsin-catalyzed hydrolysis (at pH 8.5) of methyl esters of some synthetic dipeptides containing the residues of both arginine and L(D)-p-fluorophenylalanine or L(D)-tyrosine has been studied. The digestion of Tos-(pF)Phe-Arg-OMe was shown as unfollowing the Michaelis-Menten kinetic since in the reaction course the substrate activation is observed and while the reaction product is the enzymatic process inhibitor. In contrast to this, the hydrolysis of other substrates studied, follows the normal Michaelis-Menten kinetic.

Alanine↗
PubMedDate not supplied

[Catalytic effect of gamma-thrombin on synthetic low molecular weight peptide substrates].

Hydrolysis and respective catalytic parameters of hydrolysis of ester peptide substrates that contain residues of hydrophobic and nonpolar amino acids in P2, P3 subsites have been studied. It is shown that efficiency of hydrolysis by thrombin is determined by the length of polypeptide chains and by the nature of the amino acids in P2, P3 subsites in the substrate. In spite of the fact that gamma-thrombin retains the conformation activity of the catalytic centre the local conformation changes of the second binding region of the enzyme have been discovered.

Amino Acids↗