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

Physico-chemical characteristics of urokinase in urine were studied by immunological and chemical methods. By agar zone electrophoresis, commercial urokinase preparations could be separated into an anodic and cathodic fraction. The latter reacted with urokinase antibodies with two precipitation bands. Band I displayed the major part of urokinase activity and migrated as a beta-globulin with a molecular weight of 32,000 daltons. Band II showed immunological identity with human serum, human albumin, alpha-2-macroglobulin and alpha-2-HS-glycoprotein. The specific activity of the cathodic fractions was up to 80,000 ploug units/mg protein. The ratio esterase/fibrinolytic activity did not change during the purification procedure. Further purification of the fractions with higher specific activity by affinity chromatography was unable to eliminate material cross reacting with human antisera (Band II). These findings permit the conclusion, that urokinase activity in urine is not confined to a homogeneous protein fraction. Activity is found both in a low molecular weight fraction and in a high molecular weight complex which contains serum proteins. These cannot be removed by exhaustive purification procedures and may play an important role in stabilizing and/or protecting urinary urokinase against proteolytic degradation. With Todd's technique diffuse fibrinolytic activity could be demonstrated in the kidney in the iuxtamedullary border region, (venae arcuatae, venae interlobulares, vasa recta) and in the epithelium of the calyces. Urokinase activity was specifically blocked by highly purified urokinase antibodies and could thus be distinguished from nonspecific proteolytic activity. The topographic relationship to medulla and uroepithelium may point to a role of urokinase in maintaining patency in slow flow systems.
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PMID:Isolation and renal localisation of urokinase. 10 50

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

The esterase activity of highly purified human urokinase on Nalpha-acetylglycl-L-lysine methyl ester is strongly inhibited by 1 X 10(-5) to 1 X 10(-2)M Cu++, Hg++, Ni++, Co++, Fe+++, and Mn++ solutions, whereas Na+, K+, Ca++, and Mg++ are weakly effective. This inhibition is parallel with the inhibition of activation of plasminogenby urokinase. There is no simple linear relation between inhibiton and concentration. Addition of ethylenediaminetetraacetate or electrodialysis fully reactivates the inhibited enzyme. These results are discussed in relation to similar effects of ions on trypsin.
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PMID:The effects of metal ions on esterase activities of urokinase. 41 20

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

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

The treatment of prostatic cancer with oestrogen has been reported to be associated with cardiovascular side effects. Twenty patients with recently diagnosed prostatic cancer were randomly allocated to oestrogen therapy or orchidectomy. As compared to healthy age matched controls the patients with prostatic cancer had increased base-line levels of fibrinogen (5.2 +/- 1.9 g/l versus 3.7 +/- 1.0 g/l; p less than 0.002) and factor VIII:C (166 +/- 62% versus 110 +/- 29%; p less than 0.001). During oestrogen therapy factor VII increased from 99 +/- 22% to 150 +/- 47% (p less than 0.001), while the antithrombin III level fell from 93 +/- 10% to 81 +/- 13% (p less than 0.001). Both these changes are in the direction of a hypercoaguable state. Concomitantly plasminogen increased from 113 +/- 14% to 142 +/- 18% (p less than 0.001), urokinase inhibiting activity fell from 105 +/- 10% to 90 +/- 9% (p less than 0.001) and C1-esterase inhibitor fell from 110 +/- 17% to 86 +/- 22% (p less than 0.05) in the oestrogen therapy group. After orchidectomy there were no changes in the activators and inhibitors of coagulation and fibrinolysis studied as compared to base-line values. Furthermore the D dimer, a specific degradation product of crosslinked fibrin increased from a normal to a pathological value in 4 out of 8 tested patients after 6 weeks of oestrogen therapy, but in none out of 9 tested patients in the orchidectomy group. Briefly stated, patients with prostatic cancer treated with oestrogen have increased levels of factor VII, factor VIII:C and fibrinogen and a decreased level of antithrombin III.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Activators and inhibitors of coagulation and fibrinolysis in patients with prostatic cancer treated with oestrogen or orchidectomy. 379 20

Monoclonal antibodies to purified human urinary kallikrein have been developed. Selection of antibody producing clones was based on 125I-kallikrein binding activity of hybridoma media in both radioimmunoassay and enzyme-linked immunosorbent assay. Three clones (2 IgG1, 1 IgG2b) were subcloned, characterized, and compared with the polyclonal antiserum generated in rabbits immunized with the purified kallikrein. With radioimmunoassay, mouse ascitic fluids or rabbit antisera dilutions showing 50% binding to 125I-kallikrein were 1:1.2 X 10(6) (E7A9), 1:1.2 X 10(5) (H6A6), 1:8.0 X 10(4) (E12H1), and 1:1.4 X 10(6) (the rabbit antisera). With enzyme-linked immunosorbent assay, mouse ascitic fluids from clones E7A9 and H6A6 showed half-maximal absorbance at dilutions of 1:2.1 X 10(5) and 1:1.0 X 10(5) respectively, and the polyclonal antiserum showed half-maximal absorbance at a dilution of 1:2.0 X 10(4). These monoclonal antibodies showed no cross-reactivity with rat tissue kallikrein, rat urinary plasminogen activator, or dog pancreatic kallikrein, while the polyclonal antiserum showed some cross-reactivity. The binding of monoclonal or polyclonal antibodies to 125I-human urinary kallikrein was not affected by human plasma kallikrein, thrombin, or urokinase in a competitive radioimmunoassay. By using purified human urinary kallikrein immobilized to agarose, antibodies produced by clones E7A9 and H6A6 and in the rabbit antisera were purified to homogeneity. Each of these affinity-purified antibodies inhibited the esterase activity, and two of the three inhibited the kininogenase activity, of human urinary kallikrein. A sandwich immunosorbent assay was developed to measure this kallikrein using monoclonal antibody from the clone E7A9 in conjunction with the polyclonal antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of monoclonal and polyclonal antibodies to human tissue kallikrein. 385 80

Human urine urokinase [EC 3.4.21.31] was found to be inactivated by dithiothreitol (DTT) much more severely than by 2-mercaptoethanol at the same concentration on the basis of -SH groups. Removal of DTT by dialysis restored the activities of esterase toward acetyl-glycyl-L-lysine methyl ester, plasminogen activation, and amidase toward 7-(glutaryl-glycyl-L-arginine-amido)-4-methyl coumarin. But the restoration of amidase activity was much less than that of esterase activity. The addition of DTT mediated the conversion of high molecular weight urokinase to low molecular weight urokinase, releasing several peptides. This suggests that the urokinase consists of several polypeptides linked by disulfide bonds. The molecular weight of urokinase produced with DTT was smaller than that of low molecular weight urokinase obtained by autodigestion of high molecular weight urokinase. The autodigestion was also accompanied by liberation of some peptides. But, those peptides released on autodigestion of high molecular weight urokinase were different from those appearing in the presence of DTT.
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PMID:Effect of dithiothreitol on activity and protein structure of human urine urokinase. 399 96

This study demonstrates that human plasma alpha(2)-macroglobulin preparations possess an enzymic activity that degrades fibrinogen, resulting in the formation of products whose structure resembles that of circulating fibrinogen catabolites. The sequence of degradation is similar to that observed in plasmin-catalyzed digests, in that Aalpha-chain fragmentation precedes that of Bbeta-chain. The addition of plasminogen activators to plasma induced an increase in the N-alpha-tosyl-l-arginine methyl ester HCl esterase and fibrinogenolytic activity associated with alpha(2)-macroglobulin purified from this plasma, indicating that the enzymic activity of the complex was preserved and could be increased in the presence of other plasma enzyme inhibitors. Immunochemical studies demonstrated that an alpha(2)-macroglobulin-plasmin complex had formed in urokinase-treated plasma. This alpha(2)-macroglobulin preparation manifested an esterolytic profile like that of a complex prepared from plasmin and purified alpha(2)-macroglobulin. After complex formation with alpha(2)-macroglobulin in plasma, plasmin retained less than 0.1% of its fibrinogenolytic activity. That plasmin expressed its activity while bound to alpha(2)-macroglobulin was suggested by immunoprecipitation of this activity with alpha(2)-macroglobulin antibody and by the demonstration that pancreatic trypsin inhibitor did not effectively inhibit its fibrinogenolytic or esterolytic activity. These results raise the possibility that, in addition to its activity as a major plasma proteolytic enzyme inhibitor, alpha(2)-macroglobulin may modulate enzyme-substrate interactions, such as those resulting in the formation of circulating fibrinogen catabolites, by providing a mechanism for the preservation and protection of a portion of the enzymic activity in the presence of other circulating inhibitors.
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PMID:Degradation of human fibrinogen by plasms alpha2-macroglobulin-enzyme complexes. 426 29

Urokinase-activated human plasma was studied by gel electrophoresis, gel filtration, crossed immunoelectrophoresis and electroimmunoassay with specific antibodies and by assay of esterase and protease activity of isolated fractions. Urokinase induced the formation of different components with plasminogen+plasmin antigenicity. At low concentrations of urokinase, a component with a K(D) value of 0.18 by gel filtration and post beta(1) mobility by gel electrophoresis was detected. The isolated component had no enzyme or plasminogen activity. In this plasma sample fibrinogen was not degraded for 10h, but when fibrin was formed, by addition of thrombin, fibrin was quickly lysed, and simultaneously a component with a K(D) value of 0 and alpha(2) mobility appeared, which was probably plasmin in a complex with alpha(2) macroglobulin. This complex showed both esterase and protease activity. After gel filtration with lysine buffer of the clotted and lysed plasma another two components were observed with about the same K(D) value by gel filtration as plasminogen (0.35), but beta(1) and gamma mobilities by gel electrophoresis. They appeared to be modified plasminogen molecules, and possibly plasmin with gamma mobility. Similar processes occurred without fibrin at higher urokinase concentrations. Here a relatively slow degradation of fibrinogen was correlated to the appearance of the plasmin-alpha(2) macroglobulin complex. The fibrin surface appeared to catalyse the ultimate production of active plasmin with a subsequent preferential degradation of fibrin and the formation of a plasmin-alpha(2) macroglobulin complex. The gel filtration and electrophoresis of the plasma protease inhibitors, alpha(1) antitrypsin, inter-alpha-inhibitor, antithrombin III, and C(1)-esterase inhibitor indicated that any complex between plasmin and these inhibitors was completely dissociated. The beta(1) and post beta(1) components appear to lack correlates among components occurring in purified preparations of plasminogen and plasmin.
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PMID:Different molecular forms of plasminogen and plasmin produced by urokinase in human plasma and their relation to protease inhibitors and lysis of fibrinogen and fibrin. 428 70


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