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)

Urokinase-activated human plasma was analysed by acetic acid/urea/polyacrylamide-gel electrophoresis. The bands representing plasminogen, the plasmin-alpha 2-plasmin inhibitor and plasmin-alpha 2-macroglobulin complexes were identified by immunoprecipitation with specific antibodies and by comparison with purified components. Plasminogen and the plasmin-inhibitor complexes were isolated from plasma or thrombin-clotted plasma containing 125I-labelled Glu-plasminogen (residues 1-790) and urokinase. The plasma was kept at 37 degrees C for 0.5 and 10 times the lysis time of the clotted plasma, the clotted plasma until lysis. The plasmin heavy chain from the plasmin-inhibitor complexes was subsequently prepared. Only in one case could a low-grade proteolytic conversion of Glu- forms into Lys/Met/Val-forms (residues 77-790, 68-790 and 78-790 respectively) during the preparations be detected. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and N-terminal sequence analysis of the purified plasminogen and plasmin heavy chain showed the following. The plasminogen in plasma was on the Glu- form. Glu-plasmin constituted 0.74 and 0.58 of the plasmin bound to the alpha 2-plasmin inhibitor in plasma after brief and prolonged activation respectively. The rest was Lys/Met/Val-plasmin. The clotted plasma contained about equal amounts of Glu-plasminogen and Lys/Met/Val-plasminogen, and predominantly Lys/Met/Val-plasmin complexed to alpha 2-plasmin inhibitor and alpha 2-macroglobulin. The results of the analysis of the purified material substantiated the identity of radioactive protein bands in the gel after acetic acid/urea/polyacrylamide-gel electrophoresis.
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PMID:Identification of molecular forms of plasminogen and plasmin-inhibitor complexes in urokinase-activated human plasma. 620 93

Fibrin deposition is prominent in the histopathology of a number of inflammatory lung diseases. Plasmin, activated locally in the lung, can degrade not only this fibrin but potentially structural proteins important to normal lung architecture. Because alveolar macrophages are prominent in inflammatory processes of the lung, we examined the plasminogen activator (PA) activity of human alveolar macrophages. Intact alveolar macrophages from each of 10 healthy subjects expressed PA activity. There was no difference in activity between smoking and nonsmoking individuals. The activator activity was largely cell-associated, but under certain culture conditions, macrophages released a soluble activator into the culture medium. The membrane-bound activator had an apparent molecular mass of 52-55 kD in nonreduced sodium dodecyl sulfate (SDS) gels, and monospecific antibody to urokinase neutralized the enzyme activity. Immunoprecipitation of [35S]methionine-labeled cells showed that human alveolar macrophages actually synthesize the PA in vitro. SDS-gel analysis of the immunoprecipitated material revealed the predominant species of PA to be structurally similar to reduced, active urokinase. We also examined the role of PA in the degradation of both insoluble fibrin and elastin matrices by live macrophages. Cells degraded an insoluble fibrin matrix in the presence of plasminogen whether or not the macrophages contacted the fibrin as long as proteinase inhibitors were not in the culture medium. In the presence of serum proteinase inhibitors, macrophages still degraded a fibrin matrix, but only if they were in contact with the fibrin. Live macrophages also degraded insoluble elastin only when in contact with the elastin but could do so even in the presence of serum proteinase inhibitors. In matrices containing a mixture of fibrin and elastin, cells did not degrade elastin unless plasminogen was added to the medium. These results indicate that normal alveolar macrophages synthesize and express, probably at the cell surface, a PA. The PA is physically and immunochemically similar to urokinase but is membrane bound. The PA is critical to the degradation of fibrin matrices by normal alveolar macrophages. Under tissue conditions where elastin is embedded within other structural proteins, the activator may be rate-limiting in elastin degradation as well. The findings also suggest that live macrophage proteolytic activity is relatively insensitive to the presence of serum proteinase inhibitors, suggesting a mechanism for proteolytic lung injury even in the presence of proteinase-proteinase inhibitor balance in the soluble phase.
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PMID:Degradation of fibrin and elastin by intact human alveolar macrophages in vitro. Characterization of a plasminogen activator and its role in matrix degradation. 636 89

A plasminogen activator, previously designated as rat urinary esterase A (Nustad, K., and Pierce, J. V. (1974) Biochemistry 13, 2312-2319), was separated from kallikrein of rat urine and purified to homogeneity. In polyacrylamide slab gel electrophoresis, the purified enzyme showed three closely migrating protein bands which were labeled with [14C]diisopropylphosphorofluoridate and stained on a zymogram using the chromogenic substrate methionine-alpha-naphthyl ester. Two chains, heavy chain(s) (Mr approximately 15,800, 14,200) and light chain(s) (Mr approximately 8,850, 8,550), were separated in SDS-polyacrylamide gel under reducing conditions, while two bands (Mr approximately 24,500 and 23,000) were seen under nonreducing conditions. The active site of the enzyme was associated with the heavy chain. The purified enzyme was stained for carbohydrate by the periodic acid-Schiff reagent. Five bands were distinguished in slab gel electrofocusing with isoelectric points ranging from 5.05 to 5.45. The purified enzyme lysed fibrin clots containing plasminogen but not plasminogen-free fibrin. It hydrolyzed benzyloxylcarbonyl-Gly-Gly-Arg-amino-4-trifluoromethyl coumarin, and a Km of 53 microM and a Vmax of 63 mumol/min/mg of enzyme were obtained at pH 8.0 and 37 degrees C. The enzyme cleaved kininogen substrates to produce kinin which was measured by bioassay or radioimmunoassay. The enzyme was inhibited by soybean or lima bean trypsin inhibitor, aprotinin, alpha 1-antitrypsin, phenylmethanesulfonyl fluoride, D-Phe-Phe-ArgCH2Cl, antipain, leupeptin, benzamidine, and pentamidine. Its pH optimum was 8.5 to 9.0; it was unstable on dilution and on heating. On immunoelectrophoresis, an antiserum to the esterase formed precipitin arcs with rat plasma and this enzyme at identical positions, which in turn were different from those formed with kallikrein. This urinary enzyme belongs to the family of serine proteinases and is immunologically related to urinary kallikrein.
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PMID:Purification and characterization of rat urinary esterase A, a plasminogen activator. 668 2

The complete sequence of 157 amino acids of the light (A) chain of high molecular mass urokinase from human urine was determined. The fragmentation strategy included cyanogen bromide cleavage of the S-carboxymethylated A chain at the methionine and/or tryptophan residues and use of the specific endoproteinase Lys-C. For sequence determination automated solid- or liquid-phase techniques of Edman degradation were used. C-terminal amino acids of the A chain were determined by consecutive treatment with carboxypeptidase A and B. The amino acid sequence obtained revealed a significant homology to peptide chains of other serine proteinases. Accordingly, the sequence of the A chain can be divided into three domains: 1) The growth factor domain with homologies to murine epidermal growth factor and a particular sequence of bovine clotting factor X, 2) The "kringle" domain with homologies to "kringle" structures, e.g. in plasminogen, and 3) the connecting peptide domain containing the A1 chain of low molecular mass urokinase. Together with the amino acid sequence of the B chain, which was presented by us in an earlier communication, the sequence data presented complete the primary structure of high molecular mass urokinase from human urine.
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PMID:The primary structure of high molecular mass urokinase from human urine. The complete amino acid sequence of the A chain. 675 69

The sequence of all 253 amino acids of the heavy (B-) chain of human urinary urokinase was determined. The fragmentation strategy employed included cyanogen bromide cleavage of S-carboxymethylated B-chain at Met and/or Trp residues, cleavage of acid-labile Asp-Pro bonds, and the use of the specific endoproteinases Lys-C and Arg-C for generation of overlapping fragments. For sequence determination automated solid- or liquid-phase techniques of Edman degradation were used. The amino acid sequence obtained substantiates the serine protease character of the B-chain of urokinase: a considerable homology with other serine proteinases, especially with the B-chain of human plasmin, was proved. The pertinent active site amino acids were localized: His-46, Asp-97, and Ser-198. A carbohydrate side chain, containing at least 4 glucosamine and 2 galactosamine residues, was demonstrated to be fixed at asparagine in position 144. The sequence data presented, together with the sequence of the second (A1-) chain of low molecular mass urokinase which was reported by us in an earlier communication, complete the knowledge of the whole primary structure of an active form of human urinary urokinase.
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PMID:The complete amino acid sequence of low molecular mass urokinase from human urine. 675 72

The single polypeptide chain of native plasminogen (molecular weight approx. 90000) after CNBr-cleavage and gel filtration (Sephadex G-75) yielded a high molecular weight core fraction of fragments linked by disulfide bridges and three fragments of lower molecular weight (N-terminal and C-terminal CNBr-fragments and dodecapeptide). From the reduced and S-carboxamidomethylated core fraction an additional seven fragments with molecular weights between 2000 and 38000 were obtained. The CNBr-fragments were aligned in the porcine plasminogen polypeptide chain according to sequence homologies with the known primary structure of human plasminogen. Due to the lack of two methionine residues in kringle 1 and in the N-terminal part of the light chain region and to an additional methionine residue in kringle 2 the CNBr-fragment pattern differs from that of human plasminogen. Affinity chromatography of elastase-digested, native plasminogen yielded three fragments with intact lysine binding sites, originating from the heavy chain region and a non-adsorbable fragment, corresponding to human 'mini'-plasminogen. This fragment was converted to urokinase into a proteolytically active protein which served for the isolation of the porcine plasmin light chain. With the aid of the fragments produced by the CNBr and elastase cleavage approx. 350 residues were sequenced, of which about 80% showed identity with the sequence of human plasminogen. This percentage varied depending on the region of the molecule, with the highest extent of identity (80--90%) found in the analyzed kringles 2 and 4.
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PMID:Primary structure of porcine plasminogen. Isolation and characterization of CNBr-fragments and their alignment within the polypeptide chain. 734 Dec 39

In the conversion of bovine plasminogen to bovine plasmin not only the expected urokinase-catalysed cleavage of Arg-557-Val-558, and the following autocatalytic cleavage separating the N-terminal peptide 1-77 from the heavy chain of plasmin, but also a cleavage at Arg-342-Met-343 between kringles 3 and 4 is seen. Here, kinetic studies of the interaction of bovine alpha 2-antiplasmin with bovine plasmin were performed on isolated bovine midiplasmin (lacking kringles 1-3) and on bovine plasmin containing all of the activation products from the bovine plasminogen. A series of experiments using stopped-flow fluorescence fast kinetics as well as conventional techniques suggests a reaction model in accordance with the one known for the human system. First, a tight complex (K1 in the nanomolar range) is formed in a fast reaction step; and second, a tightening of this complex occurs in a slow reaction step. The final complex is indeed so tight (Ki < or = pM), that the reaction for many practical purposes is legitimately considered irreversible. The stopped-flow method allows for the determination of reliable values of the second-order rate constant for the fast association step. At pH 7.4 and 25 degrees C, k+1 = 1.7 x 10(6) M-1 s-1 was obtained in the absence and k+1 = 0.9 x 10(6) M-1.s-1 in the presence of the kringles 1-3 domain of bovine plasmin. In contrast to this, substantial reductions of k+1 were seen in the presence of concentrations of 6-amino-hexanoic acid corresponding to lysine-binding-site interactions and far too low to be attributed to active-site interactions with the bovine plasmins (for each, Ki = 42 mM). All in all, the data indicated that the lysine-binding site(s) not of kringle 1, but of midiplasmin (those of kringles 4 and 5) are regulating the inhibition reaction.
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PMID:Stopped-flow fluorescence kinetics of bovine alpha 2-antiplasmin inhibition of bovine midiplasmin. 752 97

Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of the plasminogen activators (PAs), tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA). A library of PAI-1 mutants containing substitutions at the P1 and P1' positions was screened for functional activity against tPA and thrombin. Several PAI-1 variants that were inactive against uPA in a previous study (Sherman, P. M., Lawrence, D. A., Yang, A. Y., Vandenberg, E. T., Paielli, D., Olson, S. T., Shore, J. D., and Ginsburg, D. (1992) J. Biol. Chem. 267, 7588-7595) had significant inhibitory activity toward tPA. This set of tPA-specific PAI-1 mutants contained a wide range of amino acid substitutions at P1 including Asn, Gln, His, Ser, Thr, Leu, Met, and all the aromatic amino acids. This group of mutants also demonstrated a spectrum of substitutions at P1'. Kinetic analyses of selected variants identified P1Tyr and P1His as the most efficient tPA-specific inhibitors, with second-order rate constants (ki) of 4.0 x 10(5) M-1s-1 and 3.6 x 10(5) M-1s-1, respectively. Additional PA-specific PAI-1 variants containing substitutions at P3 through P1' were constructed. P3Tyr-P2Ser-P1Lys-P1'Trp and P3Tyr-P2Ser-P1Tyr-P1'Met had ki values of 1.7 x 10(6) M-1s-1 and 2.5 x 10(6) M-1s-1 against tPA, respectively, but both were inactive against uPA. In contrast, P2Arg-P1Lys-P1'Ala inhibited uPA 74-fold more rapidly than tPA. The mutant PAI-1 library was also screened for inhibitory activity toward thrombin in the presence and absence of the cofactor heparin. While wild-type PAI-1 and several P1Arg variants inhibited thrombin in the absence of heparin, a number of variants were thrombin inhibitors only in the presence of heparin. These results demonstrate the importance of the reactive center residues in determining PAI-1 target specificity and suggest that second sites of interaction between inhibitors and proteases can also contribute to target specificity. Finally, the PA-specific mutants described here should provide novel reagents for dissecting the physiological role of PAI-1 both in vitro and in vivo.
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PMID:Identification of tissue-type plasminogen activator-specific plasminogen activator inhibitor-1 mutants. Evidence that second sites of interaction contribute to target specificity. 772 51

Hepatocyte growth factor/scatter factor (HGF/SF) is a heparin-binding polypeptide which shares structural domains with enzymes of the blood clotting cascade. HGF/SF is secreted by cells of mesodermal origin and has powerful mitogenic, motogenic and morphogenic activity on epithelial and endothelial cells. HGF/SF is produced as a biologically inactive single-chain precursor (pro-HGF/SF) most of which is sequestered on the cell surface or bound to the extracellular matrix. Maturation into the active alpha beta heterodimer results from proteolytic cleavage by a urokinase-type protease, which acts as a pro-HGF/SF convertase. The primary determinant for receptor binding appears to be located within the alpha-chain. The interaction of the alpha-chain with the receptor is sufficient for the activation of the signal cascade involved in the motility response. However, the complete HGF/SF protein seems to be required to elicit a mitogenic response. HGF/SF binds with high affinity to a transmembrane receptor, p190MET, encoded by the MET proto-oncogene. p190MET is the prototype of a distinct subfamily of heterodimeric tyrosine kinases, including the putative receptors Ron and Sea. The mature form of p190MET is a heterodimer of two disulfide-linked subunits (alpha and beta). The alpha-subunit is extracellular and heavily glycosylated. The beta-subunit consists of an extracellular portion involved in ligand binding, a membrane spanning segment, and a cytoplasmic tyrosine kinase domain. Both subunits derive from glycosylation and proteolytic cleavage of a common precursor of 170 kDa. In polarized epithelial cells the HGF/SF receptor is selectively exposed in the basolateral plasmalemma, where it is associated with detergent-insoluble components. Two Met isoforms, carrying an intact ligand binding domain but lacking the kinase domain due to truncation of the beta-subunit, arise from alternative post-transcriptional processing of the mature form. One truncated form is soluble and released from the cells. HGF/SF binding triggers tyrosine autophosphorylation of the receptor beta-subunit. Autophosphorylation on the major phosphorylation site Y1235 upregulates the kinase activity of the receptor, increasing the Vmax of the phosphotransfer reaction. Negative regulation of the kinase activity occurs through phosphorylation of a unique serine residue (S985) located in the juxtamembrane domain of the receptor. This phosphorylation is triggered by two distinct pathways involving either protein kinase C activation or increase in intracellular Ca2+ concentration. Upon ligand binding, the HGF/SF receptor recruits and activates several cytoplasmic effectors, including phosphatidylinositol 3-kinase (PI 3-K), phospholipase C-gamma (PLC-gamma), pp60c-Src, a tyrosine phosphatase, and a Ras-guanine nucleotide exchanger.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Identification of functional domains in the hepatocyte growth factor and its receptor by molecular engineering. 776 52

Hepatocyte growth factor (HGF) is a paracrine inducer of morphogenesis and invasive growth in epithelial and endothelial cells. HGF is secreted by mesenchymal cells as an inactive precursor (pro-HGF). The crucial step for HGF activation is the extracellular hydrolysis of the Arg494-Val495 bond, which converts pro-HGF into alpha beta-HGF, the high-affinity ligand for the Met receptor. We previously reported that the urokinase-type plasminogen activator (uPA) activates pro-HGF in vitro. We now show that this is a stoichiometric reaction, and provide evidence for its occurrence in tissue culture. Activation involves the formation of a stable complex between pro-HGF and uPA. This complex was isolated from the in vitro reaction of pure uPA with recombinant pro-HGF, as well as from the membrane of target cells, after sequential addition of uPA and pro-HGF. On the cell membrane, the uPA-HGF complex was bound to the Met receptor. Monocytic cell lines, and primary monocytes after adhesion, activated efficiently pro-HGF both on their surface and in the culture medium. This activation was inhibited by anti-catalytic anti-uPA antibodies, and occurred by a stoichiometric reaction. The stoichiometry of the activation reaction suggests that the biological effects of HGF can be titrated in vivo by the level of uPA activity. Adequate amounts of uPA can be locally provided by the macrophages, which would condition the tissue microenvironment by rendering HGF bioavailable to its target cells.
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PMID:Biological activation of pro-HGF (hepatocyte growth factor) by urokinase is controlled by a stoichiometric reaction. 782 85


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