Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.73 (urokinase-type plasminogen activator)
 10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human alpha1-antitrypsin was prepared from fresh human plasma by (NH4)-SO4-precipitation, gel filtration, affinity chromatography on concanavalin A, ion exchange chromatography and isotachophoresis. Human urokinase (EC 3.4.99.26) (plasminogen activator from urine) with M, 46 000 and 36 000 was further purified from Urokinase Leo reagent preparation by gel filtration on Sephadex G-100 Superfine. The hydrolytic activity of urokinase on acetyl-glycyl-L-lysine methyl ester acetate (Ac-Gly-Lys-OMeAc) was inhibited in a strong time-dependent manner by alpha1-antitrypsin. Complex formation between enzyme and inhibitor could be demonstrated in crossed immunoelectrophoresis against anti-alpha1-antitrypsin and anti-urokinase serum as well as by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The latter method revealed the formation of 1:1 and 2:1 molar enzyme-inhibitor complexes.
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PMID:Inhibition of urokinase by complex formation with human alpha1-antitrypsin. 108 51

The absence of both nonproductive binding and substrate activation and also the good solubility of the substrates make the urokinase-catalysed hydrolysis of specific anilides a very suitable reaction for substrate structure-enzyme activity studies. Derivatives of alpha-N-acetyl-L-lysine anilide with high sigma minus-value substituents in the aniline ring were synthesized. Rate constants kappa-cat. and apparent Michaelis-Menten constants K-m (app.) are presented. From the substituent dependence of kappa-cat. and from the fact that kappa-cat. is 13 to 37 times smaller than the deacylation rate constant it is concluded that the rate-limiting step proceeds prior to deacylation. The catalytic rate constant kappa-cat. obeys a linear free-energy relationship of the Hammett type with Q equals +0.72. Two different mechanisms implied by the results obtained from the model reaction (specific base and general acid-base catalysed hydrolysis of N-acetylglycine anilides under extreme conditions) are proposed in order to account for this positive and low Q-value. In the first mechanism the breakdown of an enzyme tetrahedral intermediate is rate-limiting, while in the second one its formation controls the overall rate. The discrimination between the two mechanisms, however, could not be found.
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PMID:Structure-activity relationship in the urokinase hydrolysis of alpha-N-acetyl-L-lysine anilides. 112 14

1. Structure-activity relationships have been established for the inhibition of urokinase by aromatic diamidines. In an assay system employing purified urokinase and human plasminogen the most potent inhibitor was found in 4',4''-diamidino-2-hydroxy-1,4-diphenoxybutane which proved 5600 times more active on a molar bases than epsilon-aminocaproic acid (E-ACA). 2. 4',4''-diamidino-2-hydroxy-1,4-diphenoxybutane behaved as a competitive inhibitor of the urokinase catalyzed hydrolysis of N-alpha-acetyl-L-lysine methyl ester. At pH 7.85 and 37 degrees C the K-1 value was determined as 3.18 times 10-6 M which compares with a value of 6.79 times 10-5 M for p-aminobenzamidine and 3.57 times 10-2 M for E-ACA. 3. In two fibrinolytic tests including urokinase as activator the superiority of diamidines over E-ACA was less marked than in the pure plasminogen activation system. This was due to the presence of certain plasma proteins in the fibrinolysis assays which augmented the inhibitory strength of E-ACA. The order of effectiveness of diamidines in the lysis tests was also different from the one in the activation test. In a human fibrin clot lysis test the most active inhibitor was 3',3''-diamidino-2-hydroxy-1,4-diphenoxybutane which was 1700 times more effective on a molar basis than E-ACA. In a human plasma clot lysis test the strongest inhibitor, 2-hydroxy-stilbamidine, was 70 times more powerful than E-ACA.
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PMID:The inhibition of urokinase by aromatic diamidines. 113 21

Evidence is presented that heparin binds rabbit plasminogen types I and II under affinity chromatographic conditions using the single stage technique earlier described (Hatton, M.W.C. and Regoeczi, E. (1974) Biochim. Biophys. Acta 359, 55-65). Thus, the affinity of types I and II for Sepharose-lysine is markedly increased in the presence of heparin and elution by epsilon-aminohexanoic acid requires a steeper gradient to recover the plasminogen types. Furthermore by adding sufficient epsilon-aminohexanoic acid to non-heparinised plasma to suppress plasminogen affinity, the presence of heparin is shown to encourage binding of plasminogen (type II more so than type I) to the gel. However, the heparin effect is quickly reversed by washing the column with 0.5 M NaCl prior to elution by epsilon-aminohexanoic acid. No evidence of a stable plasminogen-heparin complex has been found from gel filtration studies and any interaction between plasminogen and heparin probably only takes place when heparin is bound to an affinity site. Studies with 35-S-labelled heparin have shown the mucopolysaccharide to bind to the free amino group of Sepharose-lysine and Sepharose-cadaverine and to be displaced by 0.5 M NaCl elution but not by 0.1 M epsilon-aminohexanoic acid. The plasminogen types produced from heparinised plasma are free from heparin and closely resemble preparations from non-heparinised plasma when compared by polyacrylamide gel electrophoresis, Sephadex gel filtration and arginine esterase activity after urokinase activation.
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PMID:The effect of heparin on the affinity chromatography of plasminogen. Demonstration of heparin-plasminogen interaction. 113 80

The two stages in the activation of human plasminogen by urokinase have been examined kinetically in order to evaluate the significance of each stage in the activation process. The cleavage of the preactivation peptide from the NH2 terminus of native plasminogen (NH2-terminal glutamic acid) is clearly catalyzed by urokinase and is the rate-limiting first step in activation (Stage 1); this reaction is 20-fold slower than the conversion of the intermediate plasminogen (NH2-terminal lysine) to plasmin (Stage 2). Both lysine and its analogoue, epsilon-aminocaproic acid, exert two effects on the activation of native plasminogen. At low concentrations of these agents, activation is greatly accelerated. Analysis of activation in the presence and absence of these agents by sodium dodecyl sulfate gel electrophoresis indicates that the activation pathway is the same in both cases with the formation of a transient intermediate plasminogen; only the kinetics of proteolysis are altered. This enhancement in the rate of activation results solely from acceleration of the Stage 1 reaction; Stage 2 is essentially unaffected at low concentrations. Stage 1 is maximally enhanced (75-fold) at either 0.0025 M epsilon-aminocaproic acid or 0.025 M lysine and occurs 4 times more rapidly than Stage 2, which becomes the rate-limiting step at these concentrations. Plasmin also cleaves the preactivation peptide from native plasminogen and this reaction rate is enhanced by the same concentrations of lysine and epsilon-aminocaproic acid. These data suggest that lysine and epsilon-aminocaproic acid, which are known to bind to plasminogen and significantly alter its conformation, may thereby enhance preactivation peptide cleavage and consequently, plasminogen activation. At high concentrations, both Stages 1 and 2 are similarly inhibited by these agents, which suggests that this effect may be exerted by the direct inhibition of urokinase. The relative rates of preactivation peptide cleavage by the enzymes urokinase, plasmin, thrombin, and ancrod were also determined. Urokinase is 10 times more effective than plasmin in catalyzing this reaction and 1.8 X 10(4) times more effective than thrombin, while ancrod does not exert an effect. No plasmin is formed by either thrombin or ancrod.
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PMID:The importance of the preactivation peptide in the two-stage mechanism of human plasminogen activation. 115 Jun 67

Plasma contained two inhibitors of plasminogen activation by urokinase when fractioned by gel filtration on Sephadex G-200. The inhibitor in the lower molecular weight fractions was separate from the principal protease inhibitors of plasma: alpha1-antitrypsin, alpha2-macroglobulin, C1 inactivator and antithrombin III, and from factor XIII. This activation inhibitor was present in both plasma and serum and its recovery was not reduced by preincubating the serum for 6 h at 37 degrees C. Its inhibitory activity was stable for several days at 4 degrees C, and was enhanced in the presence of an increased saline concentration. Preparations of the inhibitor, active in a clot lysis system, failed to inhibit the esterase activity of urokinase on N-alpha-acetyl-glycyl-L-lysine methyl ester.
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PMID:Inhibitors of plasminogen activation in human blood. 125 23

Two forms of urokinase (EC 3.4.99.26) with apparent molecular weights of 33 400 and 47 000 purified by affinity chromatography have been modified specifically with newly synthesized peptide chloroketones by affinity labeline. Rapid inactivation of the enzyme preparations was observed with Ac-Gly-Lys-CH2 Cl and Nle-Gly-Lys-CH2 Cl which might be associated with a change in which a histidine residue is lost. After performic acid oxidation, an equivalent amount of 3-carboxymethyl histidine could be recovered, indicating alkylation at the N-3 of a histidine residue. In the case of the norleucine derivative, norleucine was concomitantly incorporated into the protein. It is thus likely that urokinase belongs in the class of enzymes utilizing the Asp..His..Ser triad for their catalytic action. The two active site residues so far identified, serine and histidine, were located in the heavy chain (33 100 mol. wt) of the 47 000 molecular weight form and in the 33 400 molecular weight form, the molecular weight of which remained constant.
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PMID:Identification of an active site histidine in urokinase. 126 31

Previously, we demonstrated that the Heymann nephritis autoantigen, gp330, can serve as a receptor site for plasminogen. This binding was not significantly inhibited by the lysine analogue epsilon-amino caproic acid (EACA), indicating that plasminogen binding was not just through lysine binding sites as suggested for other plasminogen binding sites. We now report that once plasminogen is bound to gp330, it can be converted to its active form of plasmin by urokinase. This conversion of plasminogen to plasmin proceeds at a faster rate when plasminogen is first prebound to gp330. Although there is a proportional increase in the Vmax of the urokinase-catalyzed reaction with increasing gp330 concentrations, no change in Km was observed. Once activated, plasmin remains bound to gp330 in an active state capable of cleaving the chromogenic tripeptide, S-2251. The binding of plasmin to gp330 did not significantly change its enzymatic activity; however, gp330 did have a stabilizing effect on plasmin activity at 37 degrees C. While bound to gp330, plasmin is protected from inactivation by its natural inhibitor alpha 2-antiplasmin. The binding of plasmin to gp330 as analyzed by ELISA was shown to be time dependent, reversible, saturable, and specific for gp330. Inhibition of binding of both plasminogen and plasmin to gp330 by benzamidine was similar, although EACA inhibited the binding of plasmin to gp330 slightly more than the binding of plasminogen to gp330. These results indicate that the binding of plasminogen to gp330 serves as an effective means of increasing the rate of plasmin production on the glomerular and tubular epithelial cell surface while protecting the active plasmin from natural inhibitors.
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PMID:Analysis of plasmin binding and urokinase activation of plasminogen bound to the Heymann nephritis autoantigen, gp330. 128 65

The binding, internalization, and degradation of tissue-type plasminogen activator (t-PA) were studied in a rat hepatoma (Novikoff) cell line. Binding of t-PA to specific saturable high affinity binding sites (Kd = 12 nM, 54,000 sites/cell) was followed by internalization and degradation and did not require a functional active site. The catabolism of t-PA was not inhibited by an excess of urokinase-type plasminogen activator (u-PA), and t-PA bound to Novikoff membranes was not complexed to PAI-1, suggesting a mechanism independent of PAI-1. Additionally, a mannose receptor is not involved since t-PA binding was not influenced by an excess of mannose, galactose, ovalbumin, or EDTA. Furthermore, the degradation of t-PA was not influenced by 10 mM 6-aminohexanoic acid, a lysine analogue. The t-PA receptor binds to and can be eluted from wheat germ agglutinin-Sepharose. Cross-linking of t-PA with partially purified receptor and ligand blot analysis, suggest that t-PA binds to two proteins, a principal one of 55 kDa and a minor one of 43 kDa. Novikoff cells are able also to bind (Kd = 1.4 nM, 25,000 sites/cell) and degrade u-PA. The binding was inhibited by pro-u-PA and the amino-terminal fragment of u-PA, but not by an excess of t-PA. The u-PA receptor, but not the t-PA receptor, was removed by treatment with phosphatidylinositol-specific phospholipase C. Our results show that the clearance receptor for t-PA on Novikoff cells is different from the mannose receptor and the PAI-1-dependent receptor described in other cells. The rat hepatoma cells are thus a good model to study the PAI-1 independent hepatocyte-specific clearance of t-PA.
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PMID:Demonstration of a specific clearance receptor for tissue-type plasminogen activator on rat Novikoff hepatoma cells. 131 32

Basic residues Arg-118, Lys-119, Lys-128, and Arg-129 within a putative heparin-binding and receptor-binding region of the 155-amino acid form of basic fibroblast growth factor (bFGF) have been changed to neutral glutamine residues by site-directed mutagenesis of the human bFGF cDNA. The bFGF mutant (M6B-bFGF) was expressed in E. coli and purified to homogeneity. When compared to wild type bFGF, M6B-bFGF showed in cultured endothelial cells a similar receptor-binding capacity and mitogenic activity, but a reduced affinity for heparin-like low affinity binding sites, a reduced chemotactic activity, and a reduced capacity to induce the production of urokinase-type plasminogen activator. In vivo, M6B-bFGF lacked a significant angiogenic activity. Modifications of both the primary and the tertiary structure of bFGF appear to be responsible for the modified biological properties of M6B-bFGF, thus confirming the possibility to dissociate at the structural level some of the biological activities exerted by bFGF on endothelial cells.
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PMID:Structure-function relationship of basic fibroblast growth factor: site-directed mutagenesis of a putative heparin-binding and receptor-binding region. 137 64


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