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)

A plasminogen activator secreted by cultured human pancreatic carcinoma (Mia PaCa-2) cells has been purified to apparent homogeneity by procedures including Sepharose-L-arginine methyl ester affinity chromatography, Sephadex G-200 gel filtration, isoelectric focusing, and sodium dodecyl sulfate gel electrophoresis. The plasminogen activator shares many properties with urokinase including: molecular weight (55 000), isoelectric point (8.7), heat stability (60 degrees C, 30 min), PH stability (1.5-10), and its mode of activation of plasminogen. The intracellular enzyme is membrane bound and can be solubilized by detergent. Solubilized activator has a molecular weight similar to that of the secreted enzyme as determined by sodium dodecyl sulfate gel electrophoresis. The production of plasminogen activator by Mia PaCa-2 cells is totally inhibited by actinomycin D and cycloheximide.
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PMID:Purification and characterization of a plasminogen activator secreted by cultured human pancreatic carcinoma cells. 1 90

Two plasminogen activators (1 and 2) were isolated from human seminal plasma by hiigh-speed centrifugation, Sephadex-gel filtration and ion-exchange chromatography. The activators were shown to be homogeneous by polyacrylamide-disc -gel electrophoresis at pH 8.3 and 4.5, and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The molecular weights of activators 1 and 2 were estimated as 69 000 and 74 000. Their amino acid compositions are very similar, both being high in aspartic acid, glutamic acid, serine, glycine and leucine, and low in methionine, tryptophan, tyrosine, isoleucine and histidine. Activators 1 and 2 each possess 16 cysteine residues. Both activators have isoelectric points of approx. 7.0, are stable over a wide pH range at temperatures up to 60 degrees C, but lose activity at higher temperatures, particularly under very basic or acidic conditions. They are not inhibited by EDTA, Mg2+ and Ca2+ at 10 mM concentrations, but their activity decreases on addition of 10 mM-cysteine or Fe2+ and 6-aminohexanoate or sera from pregnant women. The precipitin band formed between urokinase and its antiserum is continuous with the precipitin bands formed between the seminal plasminogen activators and the urokinase antiserum. Antisera to urokinase inhibit both the activity of urokinase and the seminal plasminogen activators.
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PMID:Purification of plasminogen activators from human seminal plasma. 2 36

The binding of urokinase to human alpha2M (alpha2-macroglobulin) was investigated in comparison with the formation of the equimolar trypsin-alpha2M complex. Experiments were performed by molecular-sieving on Sephadex G-200, subunit conversion by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis after reduction and isoelectric focusing in linear sucrose gradients with ampholytes pH 3.5-10.0. Urokinase activity was determined with alpha-N-acetyl-L-lysine methyl ester and by activation of plasminogen on unheated fibrin plates. alpha2M was determined by single radial immunodiffusion. alpha2M was capable of binding some urokinase by a non-specific type of attachment that could be disrupted by isoelectric focusing but not by gel filtration. The pI of the undissociated trypsin-alpha2M complex was 6.0, and differed from that of the pure alpha2M (5.2-5.4). Likewise the pI of the immunoreactive alpha2M was 5.2 after exposure to urokinase, whereas the dissociated urokinase focused at pI 10.2. This indicated lack of true inhibitor-complex formation, which was also sustained by total absence of subunit conversion. The results are in agreement with our previous findings with pancreatic and urinary kallikreins.
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PMID:Interaction of urokinase with alpha2-macroglobulin investigated by isoelectric focusing. Evidence for non-specific dissociable binding. 7 4

Alpha2-M (alpha2-macroglobulin) was purified from human plasma by two different procedures. As well as having no detectable impurities by the usual criteria for testing the homogeneity of protein preparations, these alpha2M preparations showed a single component, after reduction in urea, of 185000 daltons by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The molecular weight of the alpha2M was found to be 718000 by sedimentation equilibrium experiments using the gravimetrically determined -v of 0.731 ml/g. The interaction of several proteinases with alpha2M was studied by using a novel discontinuous polyacrylamide-gel system, which showed clear separation of the enzyme-complexed alpha2M from the free alpha2M. These studies indicated that urokinase, as well as trypsin, chymotrypsin, plasmin and thrombin forms complexes with alphaM. The cleavage of the 185000-dalton subunit to a 85000-dalton species on interaction of trypsin with alpha2M was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after reduction of the alpha2M-trypsin complex in urea. The amino acid composition, carbohydrate content, absorption coefficient at 280 nm, the specific refractive increment and the sedimentation coefficient for these alpha2M preparations were measured. The stability of the trypsin-binding activity of the alpha2M preparations was also studied under several storage situations.
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PMID:Physical and chemical properties of human plasma alpha2-macroglobulin. 8 Feb 17

A procedure is presented for purifying a novel proteinase inhibitor in human plasma whose apparent unique biological property is to inhibit efficiently the lysis of fibrin clots induced by plasminogen activator. The final product is homogeneous as judged by disc gel electrophoresis, and immunoelectrophoresis. Its molecular weight estimated by sodium dodecyl sulfate gel electrophoresis or sedimentation equilibrium is 67,000 and 63,000, respectively. The inhibitor is a glycoprotein consisting polypeptide chain containing 11.7% carbohyrate. It migrates in the alpha2-globulin region in immunoelectrophoresis. The inhibitor is chemically and immunologically different from all the other known inhibitors in plasma. Inhibition of plasmin by the inhibitor is almost instantaneous even at 0 degrees, in contrast to the slow inhibition of urokinase (plasminogen activator in urine). Plasminogen activation by urokinase-induced clot lysis is inhibited by the inhibitor mainly through a mechanism of instantaneous inhibition of plasmin formed and not through the inhibition of urokinase. The inhibitor also inhibits trypsin. Consequently, it is suggested that this newly identified inhibitor is named alpha2-plasmin inhibitor or alpha2-proteinase inhibitor. A specific antibody directed against the inhibitor neutralizes virtually all inhibitory activity of plasma to activator-induced clot lysis. Immunochemical quantitation of the inhibitor was specific antiserum to the inhibitor and the purified inhibitor as a standard indicates that the concentration of the inhibitory in the serum of a healthy man is in or near the range of 5 to 7 mg/100 ml, which is the lowest concentration among the concentration of the proteinase inhibitors in plasma. The inhibitor and plasmin, trypsin, or urokinase form a complex which cannot be dissociated with denaturing and reducing agents. The formation of the enzyme-inhibitor complex occurs on a 1:1 molar basis and is associated with the cleavage of a unique peptide bone, which is most clearly demonstrated in the interaction of the inhibitor and beta-trypsin. In the complex formation between the inhibitor and plasmin, the inhibitor is cross-linked with the light chain which contains the active site of plasmin. It is suggested that, in a fashion analogous to complex formation between alpha1-antitrypsin and trypsin, the cross-links are formed between the active site serine of the enzyme and the newly formed COOH-terminal residue of the inhibitor, with cleavage of a peptide bond.
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PMID:Isolation and characterization of alpha2-plasmin inhibitor from human plasma. A novel proteinase inhibitor which inhibits activator-induced clot lysis. 13 98

A complex between plasmin and an inhibitor was isolated by affinity chromatography from urokinase-activated human plasma. The complex did not react with antibodies against any of the known proteinase inhibitors in plasma. A rabbit antiserum against the complex was produced. It contained antibodies agianst plasminogen+plasmin and an alpha2 protein. By crossed immunoelectrophoresis the alpha2 protein was shown to form a complex with plasmin, when generated by urokinase in plasma, and with purified plasmin. The alpha2 protein was eluted by Sephadex G-200 gel filtration with KD approx. 0.35, different from the other inhibitors of plasmin in plasma, and corresponding to an apparent relative molecular mass (Mr) of about 75000. By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Mr of the complex was found to be approx. 130000. After reduction of the complex two main bands of protein were observed, with Mr, about 72000 and 66000, probably representing an acyl-enzyme complex of plasmin-light chain and inhibitor-heavy chain, and a plasmin-heavy chain. A weak band with Mr 9000 was possibly an inhibitor-light chain. The inhibitor was partially purified and used to titrate purified plasmin of known active-site concentration. The inhibitor bound plasmin rapidly and strongly. Assuming an equimolar combining ratio, the concentration of active inhibitor in normal human plasma was estimated to be 1.1 mumol/1. A fraction about 0.3 of the antigenic inhibitor protein appeared to be functionally inactive. In plasma, plasmin is primarily bound to the inhibitor. Only after its saturation does lysis of fibrinogen and fibrin occur and a complex between plasmin and alpha2 macroglobulin appear.
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PMID:The primary inhibitor of plasmin in human plasma. 13 18

Plasminogen was found to be present in bovine milk by crossreactivity between rabbit antiserum to plasminogen and casein prepared from milk by acid precipitation. This result was further supported by recovery of intact 125I-labeled plasminogen from rabbit milk after its intravenous injection. Freshly isolated whole bovine casein was observed to undergo slow autoproteolysis at 37 degrees C. Polyacrylamide gel electrophoresis revealed gradual disappearance of major caseins accompanied by appearance and increase in intensity of numerous electrophoretic bands. This autoproteolysis was inhibited by low concentrations of epsilon-aminocaproic acid (0.1 mM) and diisopropyl fluorophosphate (1 mM); catalytic amounts of urokinase accelerated the process. Autoproteolysis of isolated bovine beta-casein was shown by both urea and sodium dodecyl sulfate gel electrophoresis to result in formation of gamma 1- and gamma 2-caseins. Similar electrophoretic bands were formed when beta-casein was degraded by plasmin prepared from bovine blood serum. These results support the hypothesis that bovine plasmin occurs in milk and is identical to alkaline milk protease.
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PMID:Plasmin-mediated proteolysis of casein in bovine milk. 15 65

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

Human antithrombin III was purified from fresh human plasma by affinity chromatography on heparin-Sepharose, affinity chromatography on concanavalin A Sepharose, gel filtration on Ultrogel AcA 34, ion exchange chromatography on DEAE A-50 Sephadex and preparative agarose gel electrophoresis. The hydrolytic activity of urokinase (plasminogen activator from urine) on acetyl-glycyl-L-lysine methylester acetate (Ac-gly-lys-OMeAc) was inhibited by antithrombin III in a slow time-dependent manner. Heparin accelerated the reaction between activator and inhibitor. Inhibition of catalytic activity was associated with the formation of an 1:1 molar complex between activator and inhibitor as revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The complex was also demonstrated by crossed immunoelectrophoresis against anti-antithrombin III.
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PMID:Inhibition of urokinase by complex formation with human antithrombin III in absence and presence of heparin. 70 90

Several molecular weight forms of plasminogen activator (PA) activity have been observed in serum-free conditioned media from human cells in culture. An antibody inhibition technique is described which combines inhibition of enzyme activity by anti-urokinase IgG with sodium dodecyl sulfate-gel electrophoresis to determine whether different molecular weight forms of human cell PA's are immunologically related to urokinase. Plasminogen activator forms with molecular weights of 85,000 to 95,000, 50,000 to 60,000, and 36,000 were inhibited by anti-urokinase IgG. In contrast, PA forms with molecular weights in the 73,000 range from three different types of human cells were not inhibited by comparable concentrations of the antibody. Human embryonic kidney cultures contain only anti-urokinase IgG-inhibitable PA forms, while melanoma-derived Malme-3M cultures contain only anti-urokinase IgG-resistant forms. Cultures of tumorigenic Detroit 562 cells and nontumorigenic IMR-90 cells contain a mixture of "antibody-sensitive" and "antibody-resistant" PA forms. The antibody-resistant 73,000-dalton PA form may be a precursor of the smaller antibody-sensitive, urokinase-related forms, or it may be the product of a second plasminogen activator gene which codes for a protein immunologically and structurally different from urokinase.
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PMID:Immunological characterization of multiple weight forms of human cell plasminogen activators. 76 81


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