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)

In order to investigate the binding of pro-urokinase (pro-UK) in urine to fibrin/Celite, the property which led to its discovery, the effect of fibrin on the plasminogen activator activity of urine was studied. The plasminogen activator activity in urine was found to be consistently about 2-fold higher when measured by fibrin plate assay than by amidolytic substrate (S-2444), when normalized against the UK reference standard. When the amidolytic activity measurement was preceded by incubation of urine with soluble fibrin, a 2-fold increase in amidolytic activity was also found. Fibrin similarly increased plasmin generation in urine enriched with Glu- or Lys-plasminogen as determined by synthetic substrate S-2251. The observed promoting effect was common to several forms of soluble fibrin and was dose dependent, whereas fibrinogen had little effect. The promoting effect of fibrin was not expressed in the presence of pro-UK or two-chain UK (TC-UK) in buffer and therefore was attributed to another constituent of urine. Since the activity was inhibited by antibodies to UK but not to t-PA, it was called fibrin activatable UK (FA-UK). Gel filtration (Sephacryl-200) of urine revealed FA-UK activity in fractions eluting at a molecular weight of approximately 100K. A 100 K band of activity was also consistently seen when concentrated urine was subjected to zymography. Treatment of concentrated urine with hydroxylamine (1 M) eradicated both these activities and was associated with an increase in baseline amidolytic activity in the urine sample indicative of the release of UK from an inhibitor complex. Moreover, passage of urine over insolubilized monoclonal antibody against UK-inhibitor (PAI-3) complexes resulted in loss of FA-UK activity and of the 100 K band on the zymogram suggesting that the complex responsible for FA-UK was related to a PAI-3 complex. Since the FA-UK activity appeared to bind to fibrin/Celite, attempts were made to investigate complexation with pro-UK. Unfortunately, due to the instability of pro-UK in urine, no reliable data were obtained. It was concluded that PAI-3 may serve as a fibrin-interacting co-factor of UK, and therefore may play a role in fibrinolysis.
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PMID:Fibrin activatable urokinase (FA-UK): a latent form of UK found in urine related to a complex with an inhibitor/fibrin-interacting cofactor. 305 14

Placental extracts contain inhibitors of human urinary urokinase. These extracts form a heterogeneous population of complexes with 125I-urokinase that are recognizable by changes in gel filtration profile and mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment with reducing agents eliminated the size heterogeneity without loss of activity, thereby allowing the placental inhibitor to be purified. Active inhibitor has been isolated in apparently homogeneous form after an eight-step procedure that included salt extraction, ammonium sulfate fractionation, column chromatography on CM-cellulose, DEAE-Sepharose, and hydroxylapatite, chromatofocusing, preparative gel electrophoresis, and hydrophobic chromatography. The purified inhibitor has Mr = 47,000. The inhibitor is relatively specific for plasminogen activators since it does not inhibit the action of plasmin, factor XIIa, plasma kallikrein, or thrombin. The inhibitor forms complexes with 1:1 stoichiometry that block the active sites of urokinase (but not prourokinase) and both one- and two-chain forms of tissue plasminogen activator. The stability of these complexes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggest that they are based on covalently bonded structures. Although both types of plasminogen activator are inhibited, the rate of interaction is significantly faster with urokinase, tissue plasminogen activator being inhibited less efficiently. The complexes formed can be dissociated by mild alkali or hydroxylamine, thereby regenerating both enzymes and inhibitor at their original molecular weights. The results suggest that the complexes are stabilized by ester-like bonds; these might involve the hydroxyl of serine at the active site of the proteases and a carboxyl group in the inhibitor.
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PMID:An inhibitor of plasminogen activation from human placenta. Purification and characterization. 310 92

We have identified a tissue-kallikrein-binding protein in human serum and in the serum-free culture media from human lung fibroblasts (WI-38) and rodent neuroblastoma X glioma hybrid cells (NG108-15). Purified and 125I-labelled tissue kallikrein and human serum form an approximately 92,000-Mr SDS-stable complex. The relative quantity of this complex-formation is measured by densitometric scanning of autoradiograms. Complex-formation between tissue kallikrein and the serum binding protein was time-dependent and detectable after 5 min incubation at 37 degrees C, with half-maximal binding at 28 min. Binding of 125I-kallikrein to kallikrein-binding protein is temperature-dependent and can be inhibited by heparin or excess unlabelled tissue kallikrein but not by plasma kallikrein, collagenase, thrombin, urokinase, alpha 1-antitrypsin or kininogens. The kallikrein-binding protein is acid- and heat-labile, as pretreatment of sera at pH 3.0 or at 60 degrees C for 30 min diminishes complex-formation. However, the formed complexes are stable to acid or 1 M-hydroxylamine treatment and can only be partially dissociated with 10 mM-NaOH. When kallikrein was inhibited by the active-site-labelling reagents phenylmethanesulphonyl fluoride or D-Phe-D-Phe-L-Arg-CH2Cl no complex-formation was observed. An endogenous approximately 92,000-Mr kallikrein-kallikrein-binding protein complex was isolated from normal human serum by using a human tissue kallikrein-agarose affinity column. These complexes were recognized by anti-(human tissue kallikrein) antibodies, but not by anti-alpha 1-antitrypsin serum, in Western-blot analyses. The results show that the kallikrein-binding protein is distinct from alpha 1-antitrypsin and is not identifiable with any of the well-characterized plasma proteinase inhibitors such as alpha 2-macroglobulin, inter-alpha-trypsin inhibitor, C1-inactivator or antithrombin III. The functional role of this kallikrein-binding protein and its impact on kallikrein activity or metabolism in vivo remain to be investigated.
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PMID:Identification of a new tissue-kallikrein-binding protein. 364 93

The complete amino acid sequence of porcine miniplasminogen (Mr 37 600), comprising 341 residues, was determined by automated Edman degradation in a liquid-phase or solid-phase sequenator. Selected fragments were produced by cleavage with 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine (BNPS-skatole), cyanogen bromide, hydroxylamine, Staphylococcus aureus protease or trypsin or with combinations thereof and by activation with urokinase. The sequence obtained was compared with the known sequences of human and bovine miniplasminogen, indicating that the porcine molecule apparently contains the same structural and functional domains as the protein of the other two species. Porcine miniplasminogen has a sequence homology of 83% with human and of 79% with bovine miniplasminogen; 74% of the amino acids are identical in all three species. The results show a higher degree of evolutionary conservatism in the structurally and/or functionally vital regions of the molecule (active site residues, kringle 5).
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PMID:Determination of the complete amino-acid sequence of porcine miniplasminogen. 384 33

Quiescent cultures of chick embryo fibroblasts incubated with human alpha-thrombin (14-219 pM) incorporated [methyl-3H]thymidine proportional to concentration. Inactivated forms of this protease (e.g. active-site-conjugated alpha-thrombin or its hirudin complex) had no mitogenic activity and did not compete with 124I-alpha-thrombin for binding to specific plasma membrane receptors. The noncoagulant but esterolytic active forms, gamma- and nitro-alpha-thrombins, were weakly mitogenic and correspondingly competed weakly for binding. Trypsin competed equally as well as native thrombin for binding, whereas chymotrypsin, elastase, and human urokinase competed with 80-fold less affinity. Plasma, arginine-specific proteases associated with nerve or epidermal growth factors, insulin, and insulin-like growth factors did not compete for binding. These data demonstrate that (a) functional catalytic residues of the thrombin active site are necessary for mitogenic activity and for specific binding; (b) regions adjacent to the active site, i.e. the high affinity protein recognition site, appear to enhance binding; and (c) the receptor can discriminate between other proteases and binds those which are also mitogens for the avian cells. The characteristics of 125I-alpha-thrombin binding were determined, and it was found to be (i) proportional to cell number; (ii) optimal at pH 6.8; (iii) 70-90% specific; (iv) at equilibrium after 60 min of incubation at 22-24 degrees C or 180 min at 0-4 degrees C (the rate constants for association, i.e. ka, at 22 and 4 degrees C were 18 and 1.1 x 10(7) M-1 min-1, respectively); and (v) essentially nondissociable. Nondissociable thrombin that bound during incubation at 0-4 degrees C was distributed equally between trypsin-sensitive and insensitive compartments. Thrombin associated with the former was released into the media when the cells were incubated at 0-4 degrees C with hirudin or hydroxylamine, or transferred to the insensitive compartment when incubated at 22 degrees C. Finally, confluent cultures of fibroblasts bind 2-3 x 10(4) 125I-alpha-thrombin molecules/cell with an apparent binding constant, i.e. Kd, of 0.7 nM (a true Kd could not be determined because of the irreversible nature of thrombin binding). The binding capacity per cell and the apparent Kd value increased proportionally to an increase in culture density.
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PMID:Protease mitogenic response of chick embryo fibroblasts and receptor binding/processing of human alpha-thrombin. 625 43

Protein C inhibitor isolated from human plasma inhibited thrombin, factor Xa, trypsin and chymotrypsin as well as activated protein C, but had very little effect on urokinase and plasmin. The inhibition constants (K1) of protein C inhibitor for activated protein C, thrombin and factor Xa were 5.6 X 10(-8) M, 6.7 X 10(-8) M and 3.1 X 10(-7) M, respectively. The second-order rate constant for inhibition of activated protein C by the inhibitor increased about 30-fold in the presence of an optimal heparin concentration (5-10 units/ml). The inhibition of activated protein C by plasma protein C inhibitor was also accelerated by heparin. When activated protein C (Mr = 62,000) was incubated with protein C inhibitor (Mr = 57,000), enzyme-inhibitor complexes with apparent Mr = 102,000 and 88,000 were observed in the nonreduced and the reduced samples, respectively, on SDS-polyacrylamide gel electrophoresis. In addition to these complexes, a band of unbound enzyme and a band with Mr = 54,000 were detected. When 125I-labeled protein C inhibitor was exposed to activated protein C, the inhibitor band was converted to bands with apparent Mr = 102,000 and 54,000 in the nonreduced samples, as determined by autoradiography after gel electrophoresis in SDS. The band with Mr = 54,000 also appeared when the inhibitor reacted with other serine proteases. The activated protein C was released from the inactive complex by treatment with 1 M ammonia or hydroxylamine. This phenomenon was found by SDS-polyacrylamide gel electrophoresis to represent the dissociation of the enzyme-inhibitor complex by ammonia or hydroxylamine into the free enzyme and the proteolytically modified inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mechanism of inhibition of activated protein C by protein C inhibitor. 632 92

Conditioned medium from cultures of human umbilical vein endothelial cells was analyzed for the presence of tissue plasminogen activator (tPA) and urokinase. Immunoprecipitation studies using metabolically labeled conditioned medium and anti-tPA IgG revealed a single band on autoradiographs corresponding to a Mr of 100,000. No bands were observed after immunoprecipitation with anti-urokinase IgG. The Mr 100,000 tPA was found to be inactive and did not bind to fibrin clots. However, exposure of this tPA to 1% NaDodSO4 resulted in the appearance of plasminogen activator activity with no apparent change in its Mr. Treatment with 10 mM diisopropylfluorophosphate prior to NaDodSO4 activation did not inhibit the NaDodSO4-induced appearance of plasminogen activator activity, indicating that the active site was not available for diisopropylfluorophosphate binding. The possibility that the properties of this Mr 100,000 tPA reflected a tPA-inhibitor complex was examined. Attempts to dissociate such a complex by denaturation, reduction, or extremes of temperature were not successful. However, after treatment of conditioned medium with 1 M hydroxylamine in the presence of 0.1% NaDodSO4, the Mr of the anti-tPA immunoprecipitable material declined by 40,000 to Mr 60,000, a Mr consistent with that of other human tPAs. Hydroxylamine has been shown previously to dissociate covalently coupled serine protease-inhibitor complexes. Furthermore, incubation of purified human melanoma cell tPA with conditioned medium resulted in an increase in its Mr by 40,000 with a concomitant decline in tPA activity. The data suggest that the latent tPA present in the conditioned medium of endothelial cells is composed of a Mr 60,000 tPA associated with an inhibitor.
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PMID:Latent tissue plasminogen activator produced by human endothelial cells in culture: evidence for an enzyme-inhibitor complex. 658 Jun 16

Addition of several arginine-specific serine proteases to culture medium conditioned by fibroblasts results in the proteases being taken into sodium dodecyl sulfate-stable complexes with a secreted factor termed protease nexin (PN) (Baker, J. B., Low, D. A., Simmer, R. L., and Cunningham, D. D. (1980) Cell 21, 37-45). PN not only inhibits these degradative enzymes but also mediates their binding, internalization, and degradation by the cells (Low, D. A., Baker, J. B., Koonce, W. C., and Cunningham, D. D. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 2340-2344). Here we describe a simple procedure for purifying milligram quantities of PN from serum-free medium conditioned by human foreskin cells. Accumulation of PN in the medium is increased by using high density microcarrier cultures supplemented with epidermal growth factor and bovine serum albumin. Application of ultrafiltration-concentrated medium to a heparin-Sepharose column followed by extensive washing of the column with buffer containing 0.2 M NaCl and elution with buffer containing 1.0 M NaCl results in the recovery of 60-90% of the input PN in a form that is 90-97% pure. This preparation can be further purified by hydrophobic chromatography on octyl-agarose. Purified PN has a molecular mass of approximately 51 kilodaltons. On nonequilibrium pH gradient electrophoresis it migrates as five bands with isoelectric points between 7.5 and 7.8. Purified PN exhibits all the properties attributed to PN in culture medium. These include: 1) formation of sodium dodecyl sulfate-stable complexes with thrombin, urokinase, and plasmin; 2) inhibition of protease activity; 3) heparin-enhanced inhibition of thrombin; and 4) cellular binding of protease-PN complexes in a heparin-sensitive reaction. When thrombin-PN complexes are dissociated with 1 M hydroxylamine a smaller form of PN (approximately 46 kilodaltons) is detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the complexed PN is proteolytically modified.
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PMID:Purification of human protease nexin. 688 87