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S A Kudinov

Publications and source records attributed to S A Kudinov.

At least 19 recordsLinked to original sources
PubMed1992

[Features of the interaction of Glu- and Lys-forms of plasminogen with native and partially hydrolyzed fibrin].

Glu- and Lys-plasminogen interaction with native and desAABB-fibrin obtained from fibrinogen partially hydrolyzed by plasmin was studied. It was found that native fibrin adsorbs 6 times more Lys-plasminogen as compared to the native form of the proenzyme. The range of the Lys-plasminogen binding does not change, if part of the fibrinogen molecules hydrolyze down to X-fragments. At the same time, the appearance in the system of 1% Xi-fragments leads to a 6-fold increase in the Glu-plasminogen binding. The amount of adsorbed Glu-plasminogen reaches the level of Lys-plasminogen adsorption both in the native and partially hydrolyzed fibrin. It was found that kringle K 1-3 or 6-aminohexanoic acid at saturating for high-affinity lysine-binding sites concentrations do not influence the Glu-plasminogen binding to native fibrin but inhibit it when the partially purified form is used. It is assumed that the manyfold increase of the Glu-plasminogen binding to partially hydrolyzed fibrin is due to the alteration of the proenzyme conformation at the initial steps of fibrin hydrolysis during the formation of Xi fragments.

Aminocaproic Acid↗
PubMed1991

[Proteolytic activity of the Glu-plasminogen complex with fibrinogen fragment E].

Glu-plasminogen interaction with fibrinogen fragment E results in the alteration of its adsorptive capacity. During this interaction in the absence of plasmin and tissue activator of plasminogen, Glu-plasminogen is transformed into a partly degraded form. Glu-plasminogen complexes with soluble and immobilized fibrinogen fragment E. contain a serine proteinase-specific activity which is inhibited by diisopropylfluorophosphate. The complexes under study are active towards fibrin and the plasmin-specific tripeptide substrate, D-Val-L-Leu-L-Lys-p-nitroanilide. It is concluded that fibrinogen fragment E induces structural changes in the enzyme molecule which eventually result in the formation of an active center.

Binding Sites↗
PubMed1990

Fluorescence spectroscopic analysis of ligand binding to kringle 1 + 2 + 3 and kringle 1 fragments from human plasminogen.

The ligand binding of kringle 1 + 2 + 3 and kringle 1 from human plasminogen has been investigated by fluorescence spectroscopy. Analysis of fluorescence titration of kringle 1 + 2 + 3 with 6-aminohexanoic acid shows that this fragment, besides the high-affinity lysine-binding site with Kd = 2.9 microM, contains two additional lysine-binding sites which differ in binding strength (Kd = 28 microM and Kd = 220 microM). This strongly suggests the existence of a lysine-binding site in each of the first three kringles. 6-Aminohexanoic acid, pentylamine, pentanoic acid and arginine were used for investigation of the ligand specificity of isolated kringle 1 prepared by pepsin hydrolysis of kringle 1 + 2 + 3. It has been established that kringle 1 has high affinity to 6-aminohexanoicacid, pentylamine and arginine (Kd values are 3.2 microM, 4.8 microM and 4.3 microM, respectively). At the same time pentanoic acid did not bind with kringle 1. These facts indicate, firstly, a broad ligand specificity of kringle 1 and, secondly, the paramount importance of the positively charged group of the ligand for its interaction with lysine-binding site of this kringle.

Amines↗
PubMed1989

Analysis of ligand binding to kringles 4 and 5 fragments from human plasminogen.

The interaction of the isolated kringles 4 and 5 from human plasminogen with 6-aminohexanoic acid, pentylamine, pentanoic acid and arginine has been quantitatively characterized by scanning calorimetry and fluorescent spectroscopy. It has been found that the ligands with the positively charged group have a good binding ability while pentanoic acid in comparison with 6-aminohexanoic acid being devoid of amino group does not interact with the kringles under study. The positively charged group of the ligand is suggested to play a crucial role in ligand binding with the lysine-binding site.

Amines↗
PubMed1989

[Binding of Glu-plasminogen by fibrinogen and byproducts of its proteolysis].

The ability of the native form of plasminogen (Glu-plasminogen) to form complexes with fibrinogen and its fragments immobilized on CNBr-agarose was studied. It was found that unlike Lys-plasminogen, the native form of the proenzyme does not bind to fibrinogen agarose. Limited proteolysis of fibrinogen by plasmin involving alpha C-domains results in the appearance of Glu-plasminogen binding sites at fibrinogen surface. The X2 fragment of fibrinogen binds to about 0.5 moles of Glu-plasminogen at an equimolar ratio of the interacting proteins. Under these conditions, the amount of bound Glu-plasminogen does not increase as a result of subsequent hydrolysis of fibrinogen down to end products, fragments E and D. It was found that Glu-plasminogen interacts with both E- and D-fragments of fibrinogen. Similar to Lys-plasminogen, Glu-plasminogen exhibits a high affinity for the E-fragment. The maximal quantity of the bound protein under the given experimental conditions is 2 moles per mole of the immobilized E-fragment. The interaction of Glu-plasminogen with the E-fragment is mediated by the lysine-binding sites of the proenzyme with a high and low affinity [Kd = 1.8.10(-6) and 7.5.10(-5) M, respectively]. Glu-plasminogen, unlike Lys-plasminogen, shows a low affinity for the D-fragment (Kd = 2.10(-5) M). Glu-plasminogen cannot be adsorbed by arginine-binding sites at the DH fragment-agarose.

Fibrin Fibrinogen Degradation Products↗
PubMed1989

[The effect of heparin on the proteolytic and fibrinolytic activity on plasmin(ogen) and fibrin clot lysis].

The effect of heparin on the proteolytic and fibrinolytic activities of plasmin and plasminogen was studied. Heparin at a concentration of 6.3.10(-6) M did not change the caseinolytic activity of plasmin and plasminogen stimulated by streptokinase but suppressed their fibrinolytic activity. At concentrations from 2.10(-8) to 0.5.10(-6) M heparin increased, whereas at 1.10(-6)-4.10(-6) M reduced the time of desAAfibrin clot half-lysis by plasmin. Within the concentration range of 2.10(-8) to 4.10(-6) M heparin did not change the time of the clot half-lysis by glu-plasminogen and slightly decreased the time of fibrin clot half-lysis by lys-plasminogen in the presence of the tissue activator. It was supposed that heparin inhibits the fibrinolytic effect of plasmin by way of formation of complexes with plasmin and reduction of plasmin specificity to the solid phase substrate, i. e., polymeric fibrin.

Animals↗
PubMed1988

[Binding of lys-plasminogen to E-fragment of fibrinogen].

The interaction of Lys-plasminogen and its fragments with fibrinogen fragment E was studied by equilibrium affinity binding. A quantitative analysis of binding parameters revealed two types of binding sites responsible for Lys-plasminogen interaction with the immobilized fragment E, i.e., with a high (Kd = 1.5 x 10(-6) M) and low (Kd = 82 x 10(-6) M) affinity ones. Among plasminogen fragments, only miniplasminogen and KI-3 bound immobilized fragment E and were eluted by epsilon-aminocaproic acid. Hence, two lysine binding sites may be involved in the binding of Lys-plasminogen to fragment E; they are localized in the KI-3 and K5 kringle structures.

Chromatography, Gel↗
PubMed1987

[Kinetics of glu- and lys-plasminogen activation by the tissue activator in a fibrin clot].

Using a modified procedure for measuring the time of fibrin clot lysis, the kinetics of Glu- and Lys-plasminogen activation by the tissue activator was studied. Within the plasminogen concentration range of 0.4-100 nM the rate of activation of both protein forms obeys the Michaelis-Menten kinetics. At Lys-plasminogen concentration equimolar to that of fibrin, the rate of activation of the former decreases down to that of Glu-plasminogen activation. The kinetic constants for Glu- and Lys-plasminogen activation (Km) are equal to 0.055 and 0.013 microM; k = 0.19 and 0.21 s-1, respectively. The Km values for fibrin-bound Glu- and Lys-plasminogen are equal to 0.25 nM and 8 nM, respectively (k = 0.08 and 0.26 s-1, respectively). It is assumed that the tissue activator exhibits a higher affinity for the Glu-plasminogen--fibrin complex than for the Lys-plasminogen-fibrin complex.

Fibrin↗
PubMed1987

[Plasminogen-binding centers of molecules of fibrinogen, fibrin and products of their proteolysis].

Using affinity chromatography, the binding of Lys-plasminogen to fibrinogen, fibrin and the consecutively formed products of their proteolysis was studied. The optimal conditions for this binding were elaborated, and the quantitative parameters of Lys-plasminogen binding to fibrinogen-Sepharose were determined. It was found that the interaction of Lys-plasminogen with fibrinogen- and fibrin-Sepharose is provided for by the lysine-binding sites of the proenzyme molecule. After partial hydrolysis of fibrinogen by plasmin, the amount of adsorbed plasminogen increases and the type of binding changes; part of the proenzyme molecules bind in the presence of 0.003 M 6-aminohexanoic acid, i.e., when lysine-binding sites appear to be blocked. A comparative study of plasminogen binding to fibrinogen fragments was carried out. The resistance of the complexes formed to the effect of 6-aminohexanoic acid and arginine competing for the binding sites was determined. The data obtained testify to the appearance of additional plasminogen-binding sites in the fibrinogen molecule during proteolysis. These sites are complementary for both lysine-and arginine-binding sites of the plasminogen molecule and are localized in the peripheral domains of the fibrinogen molecule.

Binding Sites↗
PubMed1986

Kinetic characteristics of fibrinogen and fibrin hydrolysis by plasmin 1 and 2 and miniplasmin.

Fibrinogen and fibrin hydrolysis by native plasmin 1 and 2 and by miniplasmin was studied. The degree of hydrolysis was estimated by the number of amino groups determined with trinitrobenzene sulphonic acid. The process was shown to obey Michaelis-Menten kinetics. Kinetic parameters of fibrinogen and fibrin hydrolysis by plasmin forms 1 and 2 were identical (KM = 6.5 X 10(-6) M, kcat = 7.1 sec-1) while for hydrolysis by miniplasmin KM = 20.0 X 10(-6) M, kcat = 3.58 sec-1. Thus, it was demonstrated that enzymatic properties of plasmin are to some extent dependent on the presence of lysine-binding sites. However, this appears not to have a decisive effect on fibrinolytic process.

Binding Sites↗
PubMed1986

Arginyl-binding sites of human plasminogen.

Localization and specific features of lysine- and arginyl-binding sites in Lys-plasminogen, its fragments and domains have been investigated by affinity chromatography on the sorbents containing arginine-like ligands. Lysine-binding sites of Lys-plasminogen, heavy chain and fragment K1-3 interact with the guanidyl-carboxyl pair on homoarginine-agarose. Lysine-binding site in domain K4, interacting with the amine-carboxyl pair on lysine-agarose, does not interact with that of guanidyl-carboxyl. It has been found that plasminogen contained three arginyl-binding sites interacting with guanidyl group in homoarginine-agarose. Two of them correspond to two benzamidine-binding sites in domain K5 and to the plasmin light chain while the third (unknown before) is located in fragment K1-3 and does not interact with benzamidine-agarose.

Arginine↗
PubMed1986

[Effect of fragments E and D on the plasmin hydrolysis of the fibrin clot].

It was found that fragments E (Mr = 45 000), DH (Mr = 95 000) and DL (Mr = 82 000) decrease the rate of plasmin hydrolysis of fibrin that is not cross-linked with factor XIII; the most effective inhibitor is fragment DL. The Kd values for the interactions of fragments E, DH and DL with plasmin are equal to 0.15, 0.4 and 0.04 microM, respectively.

Blood Coagulation Tests↗
PubMed1986

[Kinetic characteristics of the activation of various structural forms of plasminogen by tissue activator in the presence of fibrin].

It was shown that activation of two native plasminogen and miniplasminogen forms by the tissue activator in the presence of fibrin obeys the Michaelis-Menten kinetics. The kinetic parameters of activation of both plasminogen native forms differ insignificantly. For miniplasminogen whose molecule contains no lysine-binding sites, a marked decrease of activation power was observed. The Km value of activator for miniplasminogen is 10 times that of plasminogen form I and 20 times that of plasminogen form II. The kcat/Km value of activator for miniplasminogen is 7 times less than that of plasminogen form I and by one order of magnitude more than that of plasminogen form II. These results testify to the importance of lysine-binding sites in the native plasminogen molecule during the activation of fibrinolysis by the major physiological activator.

Fibrin↗
PubMed1984

Domains in human plasminogen.

Calorimetric studies of intramolecular melting of human plasminogen and of its fragments under various solvent conditions show that the intact plasminogen molecule consists of seven compact co-operative subunits, which can be regarded as structural domains. Five of these domains are formed by the homologous regions, the kringles, two domains are formed by the C-terminal part of the polypeptide chain that is split at activation, forming the light chain in plasmin, while the initial 76 amino acid residue peptide does not form any compact co-operative structure. The specific influence of epsilon-aminocaproic acid on the stability of the first, the fourth and, to a lesser extent, on the second kringle domain, provides evidence that these three domains in plasminogen possess lysine-binding ability. The first four kringle domains are almost independent in the molecule, while the fifth interacts with that part of the light chain not included in either of the two domains of this chain. These two domains are of different size and co-operate strongly in plasminogen, but at its activation into plasmin they decooperate and the stability of the smaller domain, which is formed by the N-terminal part of the light chain, decreases significantly. Since the light chain is responsible for the proteolytic activity of plasmin, it becomes clear that the active site of this protein is composed of two domains, as is the case for other serine proteases.

Amino Acid Sequence↗
PubMed1984

[Interaction of heavy and light chains of plasmin with fibrinogen E and D fragments].

It was demonstrated that plasminogen and the plasmin heavy chain form a complex with an immobilized fibrinogen fragment E. The E-fragment interacts, in its turn, with the immobilized heavy chain; this interaction is provided for by the lysin binding sites of the plasminogen molecule. The plasmin light chain having no lysin binding sites is specifically absorbed on the immobilized fragment D, whereas the D-fragment--on the immobilized light chain. The elution is caused by arginine or benzamidine; 6-aminohexanoic acid does not affect this interaction. It is assumed that the interaction of plasminogen and plasmin with fibrin is provided for not only by the lysine binding but also by the benzamidine binding sites of the plasminogen molecule.

Animals↗
PubMed1983

[Comparative evaluation of fibrinogen and fibrin hydrolysis with plasmin].

A high-sensitive method is developed for determining the degree of plasmin-catalyzed fibrinogen hydrolysis by the released amino groups stained with trinitrobenzene sulphoacid. The method permits determining 0.02-0.08 casein units of plasmin. The method made it possible to establish that after streptokinase activation plasmin hydrolyzes equally fibrinogen and fibrin in solution and as gel. When a tissue activator is used, fibrin intensifies significantly the plasminogen activation. Inhibition of plasmin by an inhibitor produced from soya is considerably slowed down in fibrin gel.

Animals↗
PubMed1983

[Immobilization of the human plasmin light chain and analysis of its complexes with streptokinase].

The heavy and light chains of human plasmin were separated by affinity 1-lysin-cellulose column chromatography. The S-carboxymethyl light chain derivative of human plasmin was imobilized by aminogroups by the insoluble matrices. Insoluble derivatives cf plasmin light chain retain an insignificant proteolytic activity, human plasminogen activator activity and capacity to form complexes with streptokinase. The activator activity of the immobilized light chain-streptokinase complex increases 5-10-fold with respect to the human plasminogen. When adding the light chain preparation to immobilized streptokinase its activator activity relative to the human plasminogen is twice as high. The both complexes: immobilized light chain-streptokinase and light chain-immobilized streptokinase are stable and may be reused for plasminogen activation.

Chromatography, Affinity↗