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)

Both the urokinase-type and tissue-type plasminogen activator can convert their approximately 54 kDa type-1 inhibitor (PAI-1) to an inactive form with a lower apparent molecular mass. We have determined the amino-terminal amino acid sequences of human native and converted PAI-1, and isolated PAI-1 cDNA and determined the nucleotide sequence in regions corresponding to the amino-terminus and the cleavage site. The data show that the conversion of the inhibitor consists of cleavage of an Arg-Met bond 33 residues from the carboxy-terminus, thus localizing the reactive center of the inhibitor to that position. In addition, a heterogeneity was found at the amino-terminus, with a Ser-Ala-Val-His-His form and a two-residue shorter form (Val-His-His-) occurring in approximately equal quantities.
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PMID:Plasminogen activator inhibitor type-1: reactive center and amino-terminal heterogeneity determined by protein and cDNA sequencing. 302 16

MTX peptides in which the amino acid was linked to the alpha-carboxyl group have been prepared and examined for cytotoxicity before and after treatment with proteolytic enzymes. The alanine, aspartic acid and arginine derivatives (MTX-ala, MTX-asp and MTX-arg) were synthesized by a regio-specific route, following the general procedures of Rosowsky and Montgomery. Each compound was obtained in good yield, and purity was established by TLC, HPLC, absorbance spectra and elemental analyses. The MTX peptides were not hydrolyzed by a variety of proteolytic enzymes (e.g., trypsin, plasmin, urokinase, aminopeptidase). Pancreatic carboxypeptidase A, however, hydrolyzed MTX-ala readily, MTX-asp slowly and MTX-arg not at all. The MTX-ala and, to a lesser extent, MTX-arg were substrates for pancreatic carboxypeptidase B. MTX-arg was also hydrolyzed by the endogenous carboxypeptidase N in human serum. The cytotoxicity of these MTX peptides toward L1210 cells was measured in a microculture assay system using a tetrazolium dye. MTX-ala was weakly cytotoxic (ID50 = 2.0 x 10(-6)M) compared to MTX (ID50 = 2.4 x 10(-8)M). When MTX-ala was tested in the presence of carboxypeptidase A, the ID50 value improved to 8.5 x 10(-8)M. MTX-arg gave an ID50 of 5.0 x 10(-8)M, which was not unexpected in view of its susceptibility to hydrolysis by the carboxypeptidase activity present in the fetal calf serum of the culture medium. Inclusion of carboxypeptidase B lowered the ID50 value to 2.5 x 10(-8)M. Possible clinical uses of MTX peptides are discussed.
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PMID:Chemotherapeutic potential of methotrexate peptides. 307 29

A cDNA library derived from human carcinoma cells was used to isolate a clone, pULB1000, coding for the preproenzyme form of human urokinase. This clone carries the full-length sequence coding for the signal peptide and for the A chain (157 amino acids) and B chain (253 amino acids) of urokinase in tandem. The sequence of the cDNA predicts the presence of a single lysine residue between the last amino acid of the mature A polypeptide (Phe-157) and the first amino acid of the mature B polypeptide (Ile-1). The amino acid sequence deduced from the cDNA sequence fits the published amino acid sequence data with three exceptions, the reported cysteine residue at position 131 in the A chain is a tryptophan, and glycine 366 and alanine 410 in the B chain are, respectively, a cysteine and a valine in our clone. A large Bgl I fragment (1482 bp), derived from the clone pULB1000 coding for most of the signal peptide and for the A and B chains, has been subcloned into the expression vector pCQV2. Heat induction of E. coli cells carrying the recombinant plasmid leads to the production of urokinase-like polypeptides having the expected molecular weights and being specifically recognized by antibodies raised against natural human urokinase.
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PMID:Molecular cloning, sequencing, and expression in Escherichia coli of human preprourokinase cDNA. 388 71

The presence of a peripheral zone of (presumed intracellular) plasminogen activator in the normal rabbit cornea has suggested that activator, once released, might regulate the permeability of limbal vessels and angiogenesis, by plasmin-dependent pathways. Plasminogen activator (urokinase [UK]) in rabbit serum albumin (RSA) was injected once (20 microliter, 3.7 CTA U) into the corneal stroma, 2 mm from the limbus. Sprouts arose from the engorged circumlimbal vessels (16 of 20 corneas) beginning on the third day and grew into the cornea over the next several days. Histologically, PMNs were observed in association with growing vessels. Contralateral corneas injected with UK (in RSA) previously inactivated by 99.7% with the specific active site inhibitor, Phe-Ala-Arg-chloromethyl ketone showed minimal vessel engorgement or stromal edema and no vascularization (0 to 20 corneas). Injuries to the so-called (plasminogen activator-containing)"critical zone" of the cornea which elicit neovascularization possibly do so by causing extracellular release of endogenous plasminogen activator. Thus, in addition to initiating the destructive events of ulceration, activator might initiate increases in vessel permeability and also neovascularization, which would result in the eventual arrest of ulceration.
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PMID:Plasminogen activator (urokinase) causes vascularization of the cornea. 617 46

The steady-state kinetic parameters of the tripeptides D-Val-Leu-Lys-, Ala-Phe-Lys-, and < Glu-Phe-Lys- in which the free carboxyl group was substituted with p-nitroaniline (substrate) or chloromethane (inhibitor), towards the serine proteinases plasmin (EC 3.4.21.7), thrombin (EC 3.4.21.5), urokinase, factor Xa, and trypsin (EC 3.4.21.4) were investigated. The p-nitroanilide derives were found to be very good substrates for plasmin, 2.5--40-times less efficient towards trypsin and very poor (100--10 000-times less efficient) substrates for thrombin, factor Xa and urokinase. The chloromethyl ketone derivatives were comparably efficient inhibitors of plasmin and trypsin and in general very poor (100--10 000-times weaker) inhibitors of thrombin, factor Xa and urokinase. D-Val-Leu-Lys-pNA however was a very poor substrate but D-Val-Leu-Lys-CH2Cl a very efficient inhibitor for thrombin. The variability in susceptibility of the substrates towards the enzymes was due to differences in their Michaelis constant, in their deacylation rate constant or both. the variable efficiency of the inhibitors was mostly due to differences in their dissociation constant and much less to differences in their alkylation rate constant. Only a poor correlation (r = 0.25) was found between the efficiency of the p-nitroanilides as substrate and their homologous chloromethyl ketones as inhibitor. The most notable discrepancy was observed with the D-Val-Leu-Lys derivatives towards thrombin.
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PMID:Kinetic properties of tripeptide lysyl chloromethyl ketone and lysyl p-nitroanilide derivatives towards trypsin-like serine proteinases. 644 39

The behaviour of human urokinase and porcine kidney cell plasminogen activator towards some synthetic substrates has been investigated. Although N- benzyloxycarbonylglycylglycyl -L-arginine 4-methyl-7- coumarylamide (Z-Gly-Gly-Arg-Amc) (I), glutaryl-Gly-Arg-Amc (II) and Z-Gly-Gly-Arg-Val-OMe (III) were substrates, Boc-Gly-Gly-Arg-Val-Val-Gly-Gly-OEt (IV) and Z-Ala-Pro-Gly-Arg-Val-Val-Gly-Gly-OEt (V) were neither substrates nor inhibitors. Steady-state kinetic parameters for the hydrolysis of (II) and (III) by urokinase and porcine kidney cell plasminogen activator were similar.
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PMID:The behaviour of urokinase and porcine kidney cell plasminogen activator towards some synthetic peptides. 653 12

We report the isolation of a specific protease zymogen from chicken plasma. The purification procedure involves barium citrate precipitation, ammonium sulfate fractionation, removal of plasminogen and plasmin on lysine-Sepharose, followed by anion and cation exchange, and gel permeation chromatography. Based on quantitative radioimmunoassay the zymogen is present in plasma at a concentration of 160 mg/liter, and it is obtained by our procedure in highly purified form with a yield of 1.4%. The single polypeptide chain contains an NH2-terminal alanine residue. The native molecule migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 84,000 under reducing conditions. It can be identified as an inactive proenzyme because it has very low amidolytic activity, does not react with the fluorescent active site titrant 4-methyl-lumbelliferyl p-guanidinobenzoate, and does not incorporate radioactive [3H]diisopropylfluorophosphate. It is very susceptible to limited proteolysis which converts it to an active enzyme with trypsin-like specificity. The active enzyme, likewise a single polypeptide chain, migrates as a doublet with apparent molecular weights of 39,000 and 40,000. Its amidolytic activity with synthetic peptide substrates is at least 40-fold higher than that of the proenzyme, it reacts efficiently with 4-methylumbelliferyl p-guanidinobenzoate, and incorporates [3H]diisopropylfluorophosphate while undergoing irreversible inactivation. The enzyme appears to be a reasonably efficient plasminogen activator in zymographic gels, but not in solution. With human high molecular weight kininogen as substrate the enzyme was about 25% as efficient as human plasma kallikrein. It lacks any plasminogen-independent proteolytic activity with other protein substrates, and it hydrolyzes small peptide substrates designed for both human kallikrein and urinary urokinase, respectively. Inhibition studies with peptide chloromethyl ketones indicate enzymatic properties closer to human plasma kallikrein than to the human plasminogen activator urokinase (EC 3.4.21.31). The chicken plasma enzyme and the plasminogen activator from the conditioned media of Rous sarcoma virus-transformed chick embryo fibroblasts treated with tumor promoter are different by criteria of tryptic peptide maps, and amino acid composition and enzymatic specificity. The designations chicken plasma prekallikrein plasminogen proactivator and chicken plasma kallikrein plasminogen activator are proposed for the zymogen and enzyme forms, respectively. Using rabbit antibodies against the proenzyme we developed a solid phase immunoadsorption procedure that allowed us to isolate the protein with an overall yield of 11.4%.
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PMID:A proenzyme from chicken plasma similar to human plasma prekallikrein. 655 13

Plasminogen activator inhibitor type 2 (PAI-2) is a serine proteinase inhibitor or serpin that is a major product of macrophages in response to endotoxin and inflammatory cytokines. We have explored the role of PAI-2 in apoptotic cell death initiated by tumor necrosis factor alpha (TNF). HeLa cells stably transfected with PAI-2 cDNA were protected from TNF-induced apoptosis, whereas cells transfected with antisense PAI-2 cDNA, a control gene, or the plasmid vector alone remained susceptible. The level of PAI-2 expressed by different HeLa cell clones was inversely correlated with their sensitivity to TNF. Loss of TNF sensitivity was not a result of loss of TNF receptor binding. In contrast, PAI-2 expression did not confer protection against apoptosis induced by ultraviolet or ionizing radiation. The serine proteinase urokinase-type plasminogen activator was not demonstrated to be the target of PAI-2 action. The P1-Arg amino acid residue of PAI-2 was determined to be required for protection, because cells expressing PAI-2 with an Ala in this position were not protected from TNF-mediated cell death. The results suggest that intracellular PAI-2 might be an important factor in regulating cell death in TNF-mediated inflammatory processes through inhibition of a proteinase involved in TNF-induced apoptosis.
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PMID:Plasminogen activator inhibitor type 2 inhibits tumor necrosis factor alpha-induced apoptosis. Evidence for an alternate biological function. 749 64

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

Binding parameters [association-rate (kass) and dissociation-rate (kdiss) constants, and affinity constants (KA = kass/kdiss)] for the interaction between urokinase-type plasminogen activator (u-PA) and its substrate plasminogen, its inhibitor plasminogen activator inhibitor-1 (PAI-1) and its receptor (u-PAR), were determined by real-time biospecific interaction analysis (BIA). The KA values for the binding of [S741A]recombinant plasminogen (plasminogen with N-terminal Glu and with the active site Ser741 mutagenized to Ala) or of active site-blocked plasmin (D-ValPheLysCH2-plasmin) to the 54-kDa or 32-kDa molecular forms of recombinant single-chain u-PA (rscu-PA) ranged between 0.57 x 10(6) M-1 and 1.7 x 10(6) M-1, compared to 14-22 x 10(6) M-1 for binding to the corresponding active site-blocked recombinant two-chain u-PA (rtcu-PA) moieties. KA values for binding of these plasmin(ogen) moieties to [Ser356deHAla]rtcu-PA (rtcu-PA with the active site Ser356 converted to dehydroAla) were 81 x 10(6) M-1 and 670 x 10(6) M-1, respectively. Binding of active site-blocked LMM-plasmin (a low-molecular-mass plasmin derivative lacking kringles 1-4) and of the plasmin B chain to [Ser356deHAla]rtcu-PA occurred with KA values of 3.7 x 10(6) M-1 and 0.33 x 10(6) M-1, compared to 670 x 10(6) M-1 for the binding of intact D-ValPheLysCH2-plasmin to [Ser356deHAla]rtcu-PA. The KA values for binding of latent PAI-1 to 54-kDa or 32-kDa molecular forms of rscu-PA and rtcu-PA were in the range 0.34-2.1 x 10(6) M-1. Reactivated PAI-1 bound to 54-kDa and 32-kDa rtcu-PA moieties with KA values of 26 x 10(6) M-1 and 28 x 10(6) M-1, compared to 0.77 x 10(6) M-1 and 3.2 x 10(6) M-1 for binding to the corresponding single-chain u-PA species, and 450 x 10(6) M-1 for binding to [Ser356deHAla]rtcu-PA. KA values for binding of plasmin(ogen) to the covalent rtcu-PA/PAI-1 complex were similar or somewhat higher than those for binding to uncomplexed rtcu-PA. Single-chain and two-chain 54-kDa u-PA moieties bound with a 1:1 stoichiometry and with very high affinity to u-PAR (KA of 4.6-8.5 x 10(9) M-1), whereas no significant binding of 32-kDa u-PA moieties was observed (KA < or = 0.2 x 10(6) M-1).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization of the binding of urokinase-type plasminogen activator (u-PA) to plasminogen, to plasminogen-activator inhibitor-1 and to the u-PA receptor. 792 73


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