Biomedical subjects

S D Varfolomeev

Publications and source records attributed to S D Varfolomeev.

At least 19 recordsLinked to original sources

PubMed2002

Bioinformatics and molecular modeling in chemical enzymology. Active sites of hydrolases.

Comparison and multiple alignments of amino acid sequences of a representative number of related enzymes demonstrate the existence of certain positions of amino acid residues which are permanently reproducible in all members of the whole family. The use of the bioinformatic approach revealed conservative residues in each of the related enzymes and ranked amino acid conservatism for the overall enzymatic catalysis. Glycine and aspartic acid residues were shown to be the most essential for structure and catalytic activity of enzymes. Amino acid residues forming catalytic subsite of the active site of enzymes are always highly conservative. Analysis revealed that aspartic acid carboxyl group is the most frequently employed nucleophilic (in deprotonated form) and electrophilic (in protonated form) agent involved in activation of molecules by the mechanism of general base and acidic catalyses in the catalytic sites of enzymes. Glycine is a unique amino acid possessing the highest possibilities for rotation along C-C and C-N bonds of the polypeptide chain. The conservative fixation of the glycine residue in polypeptide chains of related enzymes provides a possibility for directed assembly of amino acid residues into the catalytic subsite structure. It is possible that the conservative glycines provide known conformational mobility of the protein and the active site. Methods of molecular modeling were used for analysis of structural substitutions of conservative and non-conservative glycines and their effects on geometry of catalytic site of typical hydrolases. The substitution of glycine(s) for alanine significantly altered the catalytic site structures.

Binding Sites↗

PubMed1999

Probability description of ligand-receptor interactions. Evaluation of reliability of events with small and supersmall doses. I. Kinetics of ligand-receptor interactions.

We have developed mathematical methods for describing ligand-receptor interactions (LRI) using Markov chains. Under some conditions, the mean value of ligand-receptor complexes obtained using Markov chains coincides with that obtained from the law of mass action. Using the calculated ratio of standard deviation to mean number of ligand-receptor complexes, we show that with small concentrations of ligand-receptor complexes LRI must be described using probability methods. Using data from the literature, we show that LRI description using the mass-action law under these conditions can cause significant errors in interpretation of experimental data.

Binding Sites↗

PubMed1998

Kinetics of homeostatic behavior of receptor-enzyme systems under the action of physiologically active compounds.

We describe two types of models. In the first one it is assumed that regulation of enzyme activity occurs by the product of the enzyme reaction. In the second it is assumed that concomitant action of ligand occurs on at least two targets with opposite effects (dual-action model). Kinetic analysis of models of the first type shows that homeostatic response with respect to the key metabolite of the receptor-enzyme system is possible only if the enzyme kinetics are described by the equation of zero order. In 'dual-action' models, the homeostatic behavior of the system is defined by the mechanisms of compensation of the ligand primary effect.

Enzymes↗

PubMed1998

[Inactivation and stabilization of urokinase fibrinolytic activity in vitro].

When incubated at 30 degrees C and pH 7.4, urokinase lost fibrinolytic activity (i.e., the plasminogen-activating activity measured by the time of fibrin clot lysis) but completely retained amidase activity. The enzyme inactivation rate depended on the urokinase concentration and, at concentrations of more than 1.5 microM, was described by a second order equation, which indicated that the enzyme underwent autolytic degradation (kaut = 3.8 x 10(-3) M-1 min-1). During incubation, urokinase (54 kDa) was converted into its low-molecular-mass form (33 kDa) and products of the A-chain degradation. The amidase activity did not correlate with the fibrinolytic activity in the cases when the enzyme molecule underwent local unfolding or partial degradation. The optimum mixture of agents for stabilizing the fibrinolytic activity of urokinase was found.

Albumins↗

PubMed1996

[Kinetics of fibrin lysis by plasmin: inhibition by fibrin degradation products].

A kinetics of lysis of 125I-labeled fibrin coagulates by plasmin was studied. The strong competitive inhibition of fibrinolysis by the products of fibrin degradation was found. On the basis of the integral analysis of the complete kinetic curves of the fibrinolysis products accumulation, the Michaelis constant Km, the catalytic constant of the reaction of fibrinolysis by plasmin kcat, and the constant of inhibition by the reaction products Ki were determined to be 1.3 microM, 1.36 min-1, and 0.12 microM, respectively. The results obtained showed that the efficiency of plasmin inhibition by the reaction products exceeds that of plasmin interaction with the substrate. Thus, the process of fibrinolysis is regulated by a negative feedback mechanism.

Binding, Competitive↗

PubMed1995

Competitive inhibition of the 5-lipoxygenase-catalysed linoleate oxidation by arachidonic and 5-hydroperoxy-eicosatetraenoic acids.

Linoleic and arachidonic acids are competing substrates for 5-lipoxygenase from barley. When these two substrates are added simultaneously, arachidonic acid acts as a competitive inhibitor of linoleic acid oxidation with Ki of 20 microM, the same value as the Michaelis constant for arachidonate oxygenation by this enzyme (22 +/- 3 microM). Linoleic acid hydroperoxide accumulated in the reaction mixture does not inhibit the enzymatic process, while arachidonic acid hydroperoxy product (5-hydroperoxy-6,8,11,14-eicosatetraenoic acid) inhibits it with very low Ki equal to 0.5 microM.

Arachidonic Acid↗

PubMed1995

[Lipophilic derivatives of caffeic acid as lipoxygenase inhibitors with antioxidant properties].

We have prepared two lipophilic derivatives of caffeic acid at the carboxylic function--caffeic acid phenethyl ester, an active component of propolis, and N,N'-dicyclohexyl-O-(3,4-dihydroxycinnamoyl)-isourea. Both substances inhibit barley 5-lipoxygenase and soybean 15-lipoxygenase at micromolar concentrations. The inhibition is uncompetitive, dose-dependent and reversible. The caffeic acid derivatives also exhibit antioxidant properties and at a concentration 5-10 microM completely block the production of the reactive oxygen species in human neutrophils and in the cell-free xanthine/xanthine oxidase system.

Antioxidants↗

PubMed1995

[Synthesis of new platelet aggregation inhibitors substituted with pyridylisoxazoles and their 4,5-dihydroanalogs].

A number of substituted pyridylisoxazoles and their 4,5-dihydro analogs were synthesized by 1,3-dipolar cycloaddition of substituted nitrile oxides to either alkenes or alkynes. The synthesized compounds inhibit arachidonic acid-induced aggregation of human thrombocytes at concentrations of 10(-6) to 10(-3) M. Due to low toxicity, these compounds can be regarded as potential antithrombosis medicines.

Arachidonic Acid↗

PubMed1994

Inhibition of the respiratory burst in mouse macrophages by ultra-low doses of an opioid peptide is consistent with a possible adaptation mechanism.

The respiratory burst induced by phorbol myristate acetate in mouse macrophages was inhibited by ultra-low doses (10(-15)-10(-13) M) of an opioid peptide [D-Ala2]methionine enkephalinamide. The effect disappeared at concentrations above and below this range. The inhibition approached 50% and was statistically significant (P < 0.001). Increasing the time of the opioid incubation with cells brought about a shift in the maximal effect to lower concentrations of the opioid (from 10(-13) to 5 x 10(-15) M) and led to a decrease in the value of the effect, fully in accord with the previously proposed adaptation mechanism of the action of ultra-low doses.

Adaptation, Physiological↗

PubMed1994

Supercooperativity in platelet aggregation: substituted pyridyl isoxazoles, a new class of supercooperative platelet aggregation inhibitors.

The phenomenon of supercooperativity in platelet aggregation is manifested by the occurrence of clear-cut thresholds in dose-response relationships; in such cases the Hill coefficient has unusually high values. Approximation, by the Hill equation, of the relationship of the rate of arachidonate-induced platelet aggregation to the concentrations of either the inducer or inhibitors such as substituted pyridyl isoxazoles (synthesized by us), indomethacin, and pinane thromboxane A2, demonstrated that the Hill coefficients ranged from 30 to 100. 3-(3-Pyridyl)-5-phenylisoxazole, which exhibited maximal anti-aggregatory activity among the synthesized compounds, inhibited neither cyclooxygenase nor thromboxane synthase. The compounds affected the signal transduction pathway at/or posterior to the stage of thromboxane A2 reception.

Allosteric Regulation↗

PubMed1994

[Enzyme immunoassay of human chorionic beta-gonadotropin in biological fluids using photographic detection].

Potentialities of a new detection method making use of photographic information transformers were demonstrated as exemplified by enzyme immunoassay of human chorionic beta-gonadotropin. Commercial enzyme immunoassay kits for measurements of human chorionic beta-gonadotropin manufactured by Diaplus joint venture were used in the study. The results of traditional spectrophotometric detection and photographic detection were in good correlation, the correlation coefficient r being equal to 0.882. The sensitivity of the method permits reliable testing of clinically significant gonadotropin concentrations in biologic fluids.

Adult↗

PubMed1994

[Synthesis and biological activity of new analogs of beta-casomorphine-5].

Four new analogues of beta-cazomorphine-5 modified at the C-end with ethylenediamine- and glycine-containing derivatives were synthesized by the standard method of peptide chemistry (mixed anhydrides, carbodiimide, activated esters): H-Tyr-Pro-Phe-Pro-Gly-EtDA-Gly-II (I) H-Tyr-Pro-Phe-Pro-Gly-EtDA-Gly-Gly-II (II) H-Tyr-Pro-Phe-Pro-Gly-EtDA-Gly-Gly-CO-CH3 (III) H-Tyr-Pro-Phe-Pro-Gly-EtDA-Gly-Gly-CO-CH2-CH2-COOH (IV) The level of affinity and the degree of selectivity of the peptides towards the mu- and delta-opioid receptors of the rat brain lyophilized membranes were studied by the radioreceptor method. All the new peptides displayed analgetic activity, largely depending upon their structure.

Amino Acid Sequence↗

PubMed1994

[Inhibition of esterase by L-lysine, the activator and fibrinolytic activity of the plasmin-streptokinase activator complex].

The effect of L-lysine on some reactions catalysed by plasmin and the plasmin-streptokinase activator complex has been studied. The constants for competitive inhibition by L-lysine of the benzyloxycarbonyl-L-lysine p-nitrophenyl ester hydrolysis by the activator (Ki 116 mM), of the plasminogen activation by the activator (Ki 8 mM) and of fibrinolysis by plasmin (Ki 3 mM) were determined. It was found that L-lysine at concentrations below 0.05 M, which do not affect the activator's esterase activity, does inhibit fibrinolysis by plasmin and the activator complex. The effect of L-lysine on fibrinolysis under the action of the activator is complex: it inhibits both the activation of clot-entrapped plasminogen by the activator and lysis of fibrin by the plasmin formed. This inhibitory action of L-lysine is largely related to the fact that it lowers the sorption of the activator, plasminogen and plasmin on fibrin, competing with fibrin for their lysine-binding sites as well as worsens the activator-plasminogen binding.

Binding Sites↗

PubMed1993

Caffeic acid phenethyl ester as a lipoxygenase inhibitor with antioxidant properties.

Caffeic acid phenethyl ester, an active component of propolis extract, inhibits 5-lipoxygenase in the micromolar concentration range. The inhibition is of an uncompetitive type, i.e. the inhibitor binds to the enzyme-substrate complex but not to the free enzyme. Caffeic acid phenethyl ester also exhibits antioxidant properties. At a concentration of 10 microM, it completely blocks production of reactive oxygen species in human neutrophils and the xanthine/xanthine oxidase system.

Antioxidants↗

PubMed1993

Direct influence of morphine on the release of arachidonic acid and its metabolites.

The influence of 10(-10)-10(-6) M morphine on the release of [3H]arachidonic acid and its metabolites ([3H]AAM) from prelabeled resident peritoneal murine macrophages was investigated. Morphine enhanced [3H]AAM release from A23187- and LPS-stimulated macrophages, as well as the basal release of [3H]AAM. Dose-response curves showed a maximum at 10(-8) M morphine. Naloxone had no effect on morphine enhancement of [3H]AAM release. These results are in agreement with the hypothesis that [3H]AAM may be involved in the effects of morphine.

Animals↗

PubMed1993

Prostaglandin H synthase. Inactivation of the enzyme in the course of catalysis is accompanied by fast and dramatic changes in protein structure.

Prostaglandin H synthase (PGHS) as apo-PGHS, holo-PGHS, and holo-PGHS, inactivated in the course of catalysis was studied using chemical modification with diethyl pyrocarbonate (DEPC). The exhausted reaction with DEPC corresponded to the modification of 7 histidine residues in apo-PGHS and 4 in holo-PGHS. All 18 histidine residues became accessible for modification with DEPC in the enzyme, inactivated in the course of catalysis. The velocities of tryptic cleavage of all the three forms into two fragments were fairly different but independent of modification. Based on the results we hypothesize fast and dramatic changes in the protein structure in the course of the substrate conversion.

Animals↗

PubMed1993

[Prostaglandin H synthase. Chemical modification of histidine residues in various forms of the enzyme by diethylpyrocarbonate].

Prostaglandin H synthase (PGHS) as apo- and holoenzyme and the enzyme inactivated during the conversion of arachidonic acid into prostaglandin H2 has been modified by diethyl pyrocarbonate (DEPC). DEPC (40 mol/l mol protein) rapidly, but quantitatively differently interacted with the three forms of the enzyme (pH 6.0, 25 degrees C). The exhausted reaction with DEPC corresponded to modification of seven histidine residues in apo-PGHS and four residues in holo-PGHS. All of the 18 histidine residues were available for modification in the enzyme inactivated during the catalysis. The modification of apo-PGHS was accompanied by a concerted loss of the combined cyclooxygenase plus peroxidase and peroxidase activities. The velocities of the tryptic cleavage of the three forms of the enzyme into the 33 and 38 kDa polypeptides were essentially different, but the modification of each enzyme form did not affect the velocity of its cleavage. Two of the three histidine residues essential for the interaction with the heme within the 38 kDa fragment might be His-309 and His-388. Based on the comparison of availability for the reaction with DEPC of all the 18 histidine residues in the enzyme molecule inactivated by the interaction with arachidonic acid and on the abnormally high velocity of the tryptic cleavage of this form of PGHS, a hypothesis has been put forward about the fast and dramatic changes in the protein structure in the course of catalysis.

Catalysis↗

PubMed1992

Receptor binding on whole cells can oscillate.

The study of the kinetics of binding of the opiate receptor agonist [3H]DADLE with NG108-15 cell suspensions has revealed a new periodic biological phenomenon, i.e., oscillations of the cellular receptor activity. The absence of oscillations for binding of the receptor antagonist shows that oscillations occur as a result of the transformation of the receptor signal only.

Animals↗