Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT02079 (lysine)
 58,762 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two radiochemical esterolytic assays for urokinase are described. One assay is based on the urokinase-dependent hydrolysis of Nalpha-acetyl-glycyl-L-lysine [3H]methyl ester and the other on the urokinase-dependent activation of plasminogen and assay of generated plasmin with Nalpha-tosyl-L-arginine [3H]methyl ester. The assays are performed in tubes placed in liquid scintillation counting vials. At the end of the experiment generated [3H]methanol is extracted into the liquid scintillation cocktail and counted. Unhydrolyzed substrate largely remains in the aqueous phase and contributes only a small fraction of the counts. This facile separation of 3H-labeled alcohol from the ester substrate allows the simple and highly sensitive assay for urokinase. The assays give results in good agreement with the classical fibrin plate assay.
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PMID:Sensitive radiochemical esterolytic assays for urokinase. 0 14

Porcine plasmin (EC 3.4.21.7) is obtained from plasminogen activated by human urokinase. This enzyme can bind, in an equimolecular ratio, to an alpha2-macroglobulin isolated from porcine serum. The number of active sites of plasmin has been determined through a burst titration of nitroaniline during the presteady-state hydrolysis of an amide substrate (N-alpha-carbobenzoxy-L-arginine-p-nitroanilide). The kinetic constants relative to a very sensitive ester substrate (N-alpha-carbobenzoxy-L-lysine nitrophenylester) hydrolysis have been measured. The binding of plasmin to alpha2-macroglobulin results in a complete inhibition of proteolytic activity, a reduction of active sites number and a decrease of esterolytic activity towards this substrate. In the complex, the residual activity (about 60%) is protected from protein inhibitors. Absorbance difference spectra show that 1 mol of alpha2-macroglobulin binds 1 mol of plasmin and 2 mol of trypsin. When plasmin is first bound to alpha2-macroglobulin, only 1 mol of trypsin can gain access tothe second site without removing the plasmin, showing that a steric hindrance is implicated in the inhibition performed by alpha2-macroglobulin binding.
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PMID:[Study of the complex between porcine plasmin and alpha2-macroglobulin (author's transl)]. 5 8

Pretreatment of native plasminogen with plasmin or activators resulted in a pronounced increase in the binding of plasminogen to fibrin. The pretreated plasminogen was considered to be identical to the proteolytically degraded proenzyme with NH2-terminal lysine, valine or methionine, which is formed as an intermediate stage during activation of plasminogen. Bound plasminogen could be extracted by 6-aminohexanoic acid indicating a reversible binding between plasminogen and fibrin. Adsorption of pretreated plasminogen decreased when increasing concentrations of 6-aminohexanoic acid or trans-4-aminomethylcyclohexane-1-carboxylic acid (t-AMCHA) were present during fibrin formation. The concentration of amino acid producing a decrease in the binding of pretreated plasminogen to 0.5 of the amount bound in the absence of amino acid was 8.0-10(-5) M with 6-aminohexanoic acid and 1.7.10-5 M with t-AMCHA. The decrease in binding is most likely related to an effect of the amino acids on plasminogen, since agarose gel electrophoresis of pretreated plasminogen in the presence of 6-aminohexanoic acid or t-AMCHA showed a cathodic shift in mobility at the same range of concentrations of amino acid, which produced the decrease in binding of plasminogen to fibrin. Evidence is provided that the decrease in binding of proteolytically degraded plasminogen may result in an inhibition of fibrinolysis caused by activators.
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PMID:Differences in the binding to fibrin of native plasminogen and plasminogen modified by proteolytic degradation. Influence of omega-aminocarboxylic acids. 12 94

Interactions between tranexamic acid and protein were studied in respect of the antifibrinolytic actions of tranexamic acid. Tranexamic acid did neither show any interaction with fibrinogen or fibrin, nor was incorporated into cross-linked fibrin structure by the action of factor XIII. On the other hand, tranexamic acid bound to human plasmin with a dissociation constant of 3.5 X 10-5 M, which was very close to the inhibition constatn (3.6 X 10-5 M1 for this compound in inhibiting plasmin-induced fibrinolysis. The binding site of tranexamic acid on plasmin was not the catalytic site of plasmin, because TLCK-blocked plasmin also showed a similar affinity to tranexamic acid (the dissociation constant, 2.9-4.8 x 10-5m). in the binding studies with the highly purified plasminogen and TLCK-plasmin preparations which were obtained by affinity chromatography on lysine-substituted Sepharose, the molar binding ratio was shown to be 1.5-1.6 moles tranexamic acid per one mole protein. On the basis of these and other findings, a model for the inhibitory mechanism of tranexamic acid is presented.
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PMID:Plasminogen-plasmin system IX. Specific binding of tranexamic acid to plasmin. 12 63

A method is described by which the heavy chain of human plasmin, obtained by partial reduction of urokinase-activated plasminogen with 2-mercaptoethanol, is adsorbed on lysine coupled to polyacrylamide. The heavy chain is recovered from the adsorbent by elution with 6-aminohexanoic acid (yield 60-65%). Sulfhydryl titrations of the heavy chain showed that the partial reduction involved primarily the cleavage of the sole interchain disulfide bridge of plasmin. Dodecylsulfate-polyacrylamide electrophoresis gave essentially a single band corresponding to a component of about 60000 molecular weight. The NH2-terminal amino acid was predominantly threonine. 6-Aminohexanoic acid at different concentrations caused significant variations of the sedimentation and diffusion constants of the heavy chain indicating inhibitor-induced conformational alterations of the protein. The present results suggest that in plasmin only the heavy chain is capable of interacting with 6-aminohexanoic acid, and it appears that it is primarily this chain which plays an important role in the inhibition of the enzyme by 6-aminohexanoic acid.
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PMID:A new method of isolation and some properties of the heavy chain of human plasmin. 12 54

The reactive-site sequence of a proteinase inhibitor can be written as . . . -P3-P2-P1-P'1-P'2-P'3- . . . , where-P1-P'1-denotes the reactive site. Three semisynthetic homologues have been synthesized of the bovine trypsin-kallikrein inhibitor (Kunitz) with either arginine, phenylalanine or tryptophan in place of the reactive-site residue P1, lysine-15. These homologues correspond to gene products after mutation of the lysine 15 DNA codon to an arginine, phenylalanine or tryptophan DNA codon. Starting from native (virgin) inhibitor, reactive-site hydrolyzed, still active (modified) inhibitor was prepared by chemical and enzymic reactions. Modified inhibitor was then converted into inactive des-Lys15-inhibitor by reaction with carboxypeptidase B. Inactive des-Lys15-inhibitor was reactivated by enzymic replacement of the P1 residue according to Leary and Laskowski, Jr. The introduction of arginine was catalyzed by an inverse reaction with carboxypeptidase B, while phenylalanine or tryptophan were replaced by carboxypeptidase A. The reactivated semisynthetic inhibitors were trapped by complex formation with either trypsin or chymotrypsin. The enzyme - inhibitor complexes were subjected to kinetic-control dissociation, and the semisynthetic virgin inhibitors were isolated. The inhibitory properties of the semisynthetic inhibitors have been investigated against bovine trypsin and chymotrypsin and against porcine pancreatic kallikrein and plasmin. The homologues with either lysine or arginine in the P1 position are equally good inhibitors of trypsin, plasmin and kallikrein. The Arg-15-homologue is a slightly more effective kallikrein inhibitor than the Lys15-inhibitor. The semisynthetic phenylalanine and tryptophan homologues, however, are weak inhibitors of trypsin and still weaker inhibitors of kallikrein, but are excellent inhibitors of chymotrypsin. Their association constant with chymotrypsin is at least ten times higher than that of native Lys-15-inhibitor. A dramatic specificity change is observed with the phenylalanine and tryptophan homologues, which in contrast to the native inhibitor do not at all inhibit porcine plasmin. Thus, the nature of the P1 residue strongly influences the primary inhibitory specificity of the bovine inhibitor (Kunitz).
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PMID:Replacement of lysine by arginine, phenylalanine and tryptophan in the reactive site of the bovine trypsin-kallikrein inhibitor (Kunitz) and change of the inhibitory properties. 12 27

Affinity chromatography forms, 1 and 2, were each isolated from human Glu- and Lys-plasminogens by gradient elution from a L-lysine-substituted Sepharose column with a linear gradient of epsilon-aminocaproic acid. Although each of the two zymogen forms contains two affinity chromatography forms, the relative concentrattions of these forms in each of the zymogen preparations depended upon the plasma sample or enriched plasma fraction used for the preparation of the zymogen. Specific analytical acrylamide gel electrophoretic systems were used for the characterization of the zymogen and enzyme forms, and their component affinity chromatography forms, 1 and 2. The four zymogen affinity chromatography forms, Glu-1-plasminogen, Glu-2-plasminogen, Lys-1-plasminogen, and Lys-2-plasmingoen, show distinct stepwise differences in their molecular size and charge. The Glu-1-form is the largest in molecular size and the most acidic, and the Lys-2-form is the smallest in molecular size and the most basic. The proteolytically altered Lys-1- and Lys-2- forms appear to be specifically df the zymogen affinity chromatography forms showed a different distribution of isoelectric forms. The major isoelectric forms isolated from Glu-plasminogen with pI values of 6.2, 6.3, 6.4, and 6.6, and the major isoelectric forms isolated from Lys-plasminogen with pI values of 6.7, 7.2, 7.5, 7.8, and 8.1, (Summaria, L., Arzadon, L., Bernabe, P., Robbins, K. C., and Barlow, G. H. (1973) J. Biol. Chem. 248, 2984-2991) were shown to be mixtures of the Glu-1- and Glu-2- forms, or the Lys-1- and Lys-2- forms, respectively. Although the sialic acid contents of the Glu- and Lys- forms appear to be similar, the isolated affinity chromatography forms show distinct differences. The sialic acid contents of the Glu-1- and Lys-1- forms are identical, and are substantially higher than the sialic acid contents of the Glu-2- and Lys-2- forms which are also identical to each other. It is possible that the charge difference between the zymogen-1- and -2- forms may be related to the differences in their sialic acid content. Each of the four zymogen affinity chromatography forms, when activated by urokinase in the presence of the plasmin inhibitor, Trasylol, was converted to an apparently unique and different enzyme form. The four enzyme forms show distinct stepwise differences in molecular size; Glu-1-plasmin is the largest in size whereas Lys-2-plasmin is the smallest in size. Each plasmin-derived carboxymethyl heavy(A) chain was found to be different in molecular size, but the two carboxymethyl light(B) chains found in each of the four enzyme forms appeared to be identical and of the same molecular sizes. The four heavy(A) chains show a stepwise difference in molecular size; the Glu-1-heavy(A) chain is the largest in size whereas the Lys-2-heavy(A) chain is the smallest in size...
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PMID:Isolation and characterization of the affinity chromatography forms of human Glu- and Lys-plasminogens and plasmins. 13 40

A functionally active human plasmin light (B) chain derivative, stabilized by the streptomyces plasmin inhibitor leupeptin, was isolated from a partially reduced and alkylated enzyme preparation by an affinity chromatography method with a L-lysine-substituted Sepharose column. This light (B) chain derivative was found to be relatively homogeneous by electrophoretic analysis in both an acrylamide gel/dodecyl sulfate system and on cellulose acetate. It possessed approximately 3% of the proteolytic activity (casein substrate) of the original enzyme, and it incorporated 0.09 mol of [3H]diisopropyl phosphorofluoridate per mol of protein. It contained 3.1 +/- 0.3 carboxymethylated cysteines per mol of protein and can be designated as a CmCys5-light (B) chain (CmCys)3. When this isolated light (B) chain derivative was mixed in equal molar amounts with streptokinase, the mixture developed both human and bovine plasminogen activator activities; the bovine activator activity was approximately 66% of the bovine activator activity of the equimolar human plasmin-streptokinase complex. Although this complex now incorporated 0.50 mol of [3H]diisopropyl phosphorofluoridate per mol of protein, its proteolytic activity, on a molar basis, was the same as the proteolytic activity of the isolated light (B) chain derivative. It was shown by electrophoretic analysis in both an acrylamide gel/epsilon-aminocaproic acid system and on cellulose acetate that the light (B) chain derivative and streptokinase forms an equimolar light (B) chain-streptokinase complex, indicating that the binding site for streptokinase is located on the light (B) chain of the enzyme. A functionally active equimolar light (B) chain-streptokinase complex was also isolated from a partially reduced and alkylated equimolar human plasmin-streptokinase complex by the affinity chromatography method. The plasminogen activator activities (human and bovine) of this light (B) chain-streptokinase complex were similar to those of the plasmin-streptokinase complex from which it was derived. Although this complex incorporated 0.70 mol of [3H]diisopropyl phosphorofluoridate per mol of protein, its proteolytic activity, on a molar basis, was only 14% of proteolytic activity of the plasmin-streptokinase complex.
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PMID:Isolation of a human plasmin-derived, functionally active, light (B) chain capable of forming with streptokinase an equimolar light (B) chain-streptokinase complex with plasminogen activator activity. 13 97

The effects of a single 1-min extraction with chloroform (CHCl3) on plasma fibrinolytic activity has been examined by 125I-fibrin solid phase assay, using normal plasma and plasma depleted of plasminogen (PLG) by lysine-Sepharose affinity chromatography. Fibrinolytic activity of normal plasma is increased (40%-175%), and more than 95% of antiplasmin activity is removed. The increase is demonstrable in PLG-depleted plasma, and is not inhibited by tranexamic acid (0.01 M). Purified PLG is not activated to plasmin by ChCl3 treatment. Bio-Gel A 0.5 m fractionation of CHCl3-extracted, PLG-depleted plasma reveals fractions with the following activities: (1) streptokinase-activatable, PLG-independent fibrinolytic activities; (2) PLG activator activities; and (3) plasmin-stimulated but PLG-independent fibrinolytic activities, which include activities inhibited by hexadimethrine bromide and which cofractionate in part with plasmin-stimulated procoagulant activities. In addition, similar fractionation of nonextracted plasma reveals two non-plasmin fibrinolytic activities (approximately 30,000 and 13,000 daltons) activated by streptokinase and plasmin, respectively. The findings indicate that the enhanced fibrinolytic activity resulting from CHCl3 treatment is independent of plasmin as the ultimate fibrinolytic enzyme, although activities stimulated by plasmin may contribute, and that such treatment is a useful maneuver for study of PLG-dependent and PLG-independent fibrinolytic mechanisms, and their interactions.
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PMID:Mechanisms involved in enhancement of plasma fibrinolytic activity by chloroform. 13 88

Fresh plasma was seeded with trace amounts of highly purified biologically intact iodine-labelled plasminogen and the plasmin-inhibitor complexes formed after activation with streptokinase or urokinase separated by gel filtration. Two radioactive peaks were observed, the first one eluted in the void volume and the second one just before the 7-S globulin peak. In incompletely activated samples, the second peak was always predominant over the first one. Both components were purified with high yield by a combination of affinity chromatography on lysine-agarose and gel filtration, and investigated by dodecylsulphate-polyacrylamide gel electrophoresis and immunoelectrophoresis. Neither component reacted with antisera against alpha1-antitrypsin, antithrombin III, C1-esterase inhibitor, inter-alpha-trypsin inhibitor or alpha1-antichymotrypsin. The component of the first peak appeared to be a complex between plasmin and alpha2-macroglobulin which reacted with antisera against human plasminogen and against alpha2-macroglobulin. The component of the second peak had a molecular weight (Mr) of 120000-140000 by dodecyl-sulphate-polyacrylamide gel electrophoresis and lpon reduction displayed a doublet band with an Mr of 65000-70000 and a band with Mr 11000. It reacted with antisera against plasminogen and with antisera raised against this complex and absorbed with purified plasminogen. The latter antisera reacted with a single component in plasma which is different from the above-mentioned plasma protease inhibitors. Specific removal of this component from plasma by immuno-absorption resulted in disappearance of the fast-reacting antiplasmin activity whereas alpha2-macroglobulin was found to represent the slower-reacting plasmin-neutralizing activity. In the presence of normal plasma levels of these proteins, the specific removal or absence of alpha1-antitrypsin, antithrombin III or C1-esterase inhibitor did not alter the inactivation rate of plasmin when added to plasma in quimolar amounts to that of plasminogen. It is concluded that only two plasma proteins are important in the binding of plasmin generated by activation of the plasma plasminogen, namely a fast-reacting inhibitor which is different from the known plasma protease inhibitors and which we have provisionally named antiplasmin, and alpha2-macroglobulin, which reacts more slowly.
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PMID:Identification and some properties of a new fast-reacting plasmin inhibitor in human plasma. 13 45


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