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)

The synthetic thrombin-inhibitor termed No. 205 (N-alpha-dansyl-L-arginine-4-ethyl-piperidine amide) found in our laboratories was studied kinetically using synthetic peptide substrates. The following results were obtained. 1. No. 205 inhibited thrombin competively with bz-Phe-Val-Arg-pNA and the Ki value obtained was extremely small, 3.7 x 10(-8) M. 2. No. 205 also inhibited trypsin competitively with bz-Phe-Val-Arg-pNA but the Ki value obtained was far larger than that for thrombin, 1.0 x 10(-5) M. 3. No. 205 inhibited F. Xa, plasmin and urokinase only to a small extent when estimated using 2 x 10(-4) M D-Val-Leu-Lys-pNA, bz-Ile-Glu-Gly-Arg-pNA and Glu-Gly-Arg-pNA, respectively. 4. No 205 differed from APPA in its specific inhibitory spectrum for thrombin as compared to trypsin, plasmin and F. Xa. The above results indicate that No. 205 is an extremely potent and highly selective reversible thrombin-inhibitor.
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PMID:Kinetic studies on the selectivity of a synthetic thrombin-inhibitor using synthetic peptide substrates. 15 13

Pro-Gly-ArgCH2Cl, a reagent corresponding to the C-terminal sequence generated in plasminogen on activation by urokinase (EC 3.4.99.26) and probably by other plasminogen activators, was prepared. Pro-Gly-ArgCH2Cl was effective in the inactivation of urokinase at the 10(-6) M level (Ki 68 micrometers and k2 0.47 min-1). In contrast, only a slow inactivation was obtained by 10(-2) M N-tosyllysine chloromethyl ketone. Glu-Gly-ArgCH2Cl, N,N-dimethylaminonaphthalene-5-sulfonyl-Glu-Gly-ArgCH2Cl, and Ac-Gly-Gly-ArgCH2Cl were more reactive than Pro-Gly-ArgCH2Cl against urokinase by factors of 25, 6, and 3, respectively. The effectiveness of arginine chloromethyl ketones as affinity labels is highly dependent on binding in the S2 and S3 sites, thus sequence variations in the reagents exhibited differences in reactivity of up to four orders of magnitude. The most effective reagents had Gly in P2. Ac-Gly-Gly-ArgCH2Cl inactivates urokinase 50 times more rapidly than it does plasmin, thus providing a means of distinguishing the activity of plasmin from its activating protease whereas urokinase is almost inert to Ala-Phe-LysCH2Cl, a reagent which inactivates plasmin at the 10(-7) M level.
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PMID:The susceptibility of urokinase to affinity labeling by peptides of arginine chloromethyl ketone. 46 6

Twenty peptide-4-methylcoumarin amides (MCA) were newly synthesized and tested as possible substrates for alpha-thrombin, factor Xa, kallikreins, urokinase, and plasmin. These fluorogenic peptides contained arginine-MCA as the carboxyl-terminus. Release of 7-amino-4-methylcoumarin was determined fluorometrically. Of these peptides, the following were found to be specific substrates for individual enzymes: Boc-Val-Pro-Arg-MCA for alpha-thrombin, Boc-Ile-Glu-Gly-Arg-MCA, and Boc-Ser-Gly-Arg-MCA for factor Xa, Z-Phe-Arg-MCA for plasma kallikrein, Pro-Phe-Arg-MCA for pancreatic and urinary kallikreins, and glutaryl-Gly-Arg-MCA for urokinase. Moreover, these peptide-MCA substrates were resistant to plasmin.
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PMID:New fluorogenic substrates for alpha-thrombin, factor Xa, kallikreins, and urokinase. 59 14

Synthetic procedures have been developed for the preparation of peptides of arginine chloromethyl ketone and applied in the preparation of affinity labels which correspond to the -Pro-Phe-Arg- C terminus of bradykinin, a physiological cleavage site of kallikrein in kininogen. Two such reagents, Ala-Phe-ArgCH2C1 and Pro-Phe-ArgCH2C1, proved to be highly effective as well as selective affinity labels for human plasma kallikrein. For example, Pro-Phe-ArgCH2C1 inactivates plasma kallikrein 50% in 24 min at a concentration of 2 x 10(-8)M, while other trypsin-like proteases are less susceptible in inactivation than kallikrein, differing by a factor of 48 for plasmin and factors of 10(2)-10(5) for factor Xa, thrombin, and urokinase. The affinity of human plasma kallikrein for Ala-Phe-ArgCH2C1 (Ki = 0.078 micron) is about 60 times that for Ala-Phe-LysCH2C1(Ki = 4.9 micron), whereas human plasmin exhibits about the same affinity for the former affinity label (Ki = 1.3 micron) as for the latter (Ki = 0.83 micron). The rate constants for the irreversible step of the affinity labeling reaction, k2, are similar for affinity labels tested with the individual proteases: 0.35 min-1 for plasma kallikrein and 0.18 min-1 for plasmin.
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PMID:Synthesis of peptides of arginine chloromethyl ketone. Selective inactivation of human plasma kallikrein. 72 86

The urokinase-type plasminogen activator receptor (u-PAR) was demonstrated on cultured smooth muscle cells (SMCs) of bovine aorta. Binding of 125I-urokinase-type plasminogen activator (u-PA) was concentration dependent and saturable within 45-60 minutes. A similar concentration and time dependence was found in functional plasminogen activation studies. Human two-chain high-molecular-weight u-PA and its proenzyme (pro-u-PA) bound specifically with identical affinity (Kd). Activation of pro-u-PA was strongly accelerated on binding to SMCs and occurred only in the presence of plasminogen on the cell surface. A 100-fold molar excess of unlabeled high-molecular-weight u-PA effectively blocked binding of the radiolabeled ligands; tissue-type plasminogen activator, plasminogen, low-molecular-weight u-PA, and unrelated proteins did not. 125I-u-PA binding was abolished by a monoclonal antibody against the specific u-PA sequence responsible for u-PAR binding. Binding of u-PA sharply decreased on SMC exposure to phosphatidylinositol-specific phospholipase C, confirming the glycan phospholipid cell anchorage of u-PAR. Bovine and human alpha-thrombin (240 nM) increased the binding of 125I-u-PA fivefold, translating into an increase in the number of sites per cell from about 10(5) to 5 x 10(5) without significant change in the Kd (1.29 +/- 0.39 nM). Active site blockade of thrombin by D-Phe-Pro-Arg-chloromethyl ketone resulted in the total loss of stimulatory activity, as did the use of the inactive active site thrombin mutant, S205A. Hirugen (100 microM), which blocks the anion-binding exosite of thrombin, blocked u-PAR stimulating activity. Thus, both the catalytic activity and integrity of the exosite are important for thrombin's stimulatory activity. Other SMC mitogens (epidermal growth factor, transforming growth factor-beta 1, basic fibroblast growth factor, platelet-derived growth factor, and phorbol 12-myristate 13-acetate) increased u-PAR expression on SMCs six- to 20-fold while concomitantly increasing Kd four- to 10-fold. In all cases the induction of u-PAR was dependent on de novo protein synthesis. These observations assign a possible role for thrombin and other mitogens in u-PAR regulation, thereby influencing the pericellular proteolysis that is important in SMC migration and atheromatous plaque development.
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PMID:Regulation of the urokinase-type plasminogen activator receptor on vascular smooth muscle cells is under the control of thrombin and other mitogens. 132 97

The role of glycosylation on the enzymatic properties of single chain urokinase-type plasminogen activator (scu-PA) was investigated by site-specific mutagenesis of the glycosylated Asn-302 residu to Gln. In addition, the role of the NH2-terminal polypeptide chain and of the Cys-148 to Cys-279 interchain disulphide bond on the activity of non-glycosylated scu-PA was investigated. Therefore, variants of recombinant scu-PA (rscu-PA) were produced by transfecting Chinese hamster ovary cells with cDNA encoding rscu-PA N302Q (rscu-PA with Asn-302 to Gln mutation), rscu-PA C279A,N302Q (rscu-PA with Cys-279 to Ala and Asn-302 to Gln mutations) or rscu-PA del(N2-F157)C279A,N302Q (rscu-PA C279A,N302Q with deletion of Asn-2 through Phe-157). These mutants were purified to homogeneity from conditioned cell culture medium and were obtained essentially as single chain molecules with specific activities on fibrin plates of (mean +/- S.E.; n = 6) 45,000 +/- 5000. IU/mg, 19,000 +/- 800 IU/mg and < or = 100 IU/mg for rscu-PA N302Q, rscu-PA C279A,N302Q and rscu-PA del(N2-F157)C279A,N302Q, respectively, as compared to 64,000 +/- 2600 IU/mg for wild-type rscu-PA obtained in the same expression system. Plasmin quantitatively converts rscu-PA N302Q and rscu-PA C279A,N302Q to amidolytically active two-chain derivatives with a specific activity of 56,000 IU/mg and 32,000 IU/mg, respectively, as compared to 75,000 IU/mg for wild-type rscu-PA. Plasminogen activation as a function of time was comparable for rscu-PA N302Q and wild-type rscu-PA, and somewhat slower for rscu-PA C279A,N302Q. In a human plasma milieu in vitro, consisting of a 125I-fibrin labeled plasma clot submerged in plasma, 50 percent clot lysis in 2 h required 2.2 micrograms/ml rscu-PA N302Q and 6.0 micrograms/ml rscu-PA C279A,N302Q, as compared to 3.2 micrograms/ml wild-type rscu-PA. In contrast, rscu-PA del(N2-F157)C279A,N302Q was not converted to an amidolytically active two chain derivative by plasmin, and did not induce significant plasminogen activation in purified systems or clot lysis in a human plasma milieu. Following bolus injections in hamsters, the initial half-lives (1.8-2.6 min) and the plasma clearances (0.6-1.5 ml min-1) were comparable for wild-type rscu-PA and for the three rscu-PA mutants. These results suggest that the fibrinolytic activity in a plasma milieu in vitro and the in vivo turnover of rscu-PA are not markedly affected by the absence of carbohydrate.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Biochemical properties of recombinant mutants of nonglycosylated single chain urokinase-type plasminogen activator. 139 Sep 10

Based on studies of structure-activity relationship, trans-4-aminomethylcyclohexanecarbonyl-L-phenylalanine-4-carbox ymethylanilide (Tra-Phe-APAA) was designed as a selective plasma kallikrein inhibitor and synthesized. Tra-Phe-APAA inhibited plasma kallikrein with a Ki value of 0.81 microM, while it inhibited glandular kallikrein, plasmin, urokinase, factor Xa and thrombin with Ki values of greater than 500, 390, 200, greater than 500, and greater than 500 microM, respectively. However, its stereoisomer, Tra-D-Phe-APPA did not exhibit any detectable inhibitory activity against the above enzymes.
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PMID:Synthesis of trans-4-aminomethylcyclohexanecarbonyl-L- and -D-phenylalanine-4-carboxymethylanilide and examination of their inhibitory activity against plasma kallikrein. 139 97

Ketomethylene pseudopeptide analogues Aa-Pro-Arg psi (COCH2) Gly-pip, 1, where Aa are D- or L-amino acids (Dpa, beta, beta-diphenylalanine; alpha Nal, alpha-naphthylalanine; beta Nal, beta-naphthylalanine; Fgl, fluorenylglycine) with highly lipophilic side chains and psi (COCH2) is a ketomethylene pseudopeptide bond, have been synthesized through a modified Dakin-West reaction under very mild conditions with a high yield using tripeptide 4 with a labile functional group directly on the side chain. Their enzymatic assay of thrombin inhibition has been carried out. The structure-activity relationship study indicated that a lipophilic side chain on the amino acid in the P3 position is very important for binding to the apolar site of thrombin. Compound 1a with D-Dpa at the P3 position has a Ki of 0.2 microM and it doubles thrombin clotting time at only 3 times higher concentration. These values are about 7 times better than those of the corresponding D-Phe analogues. Furthermore, 1a shows poor inhibitory activity against plasmin, factor Xa, urokinase, and kallikrein. Preliminary in vivo testing (3-4-kg rabbit as the animal model) shows no observable side effect (change of blood pressure and accumulation of blood platelet in lungs) at a dose of 1 mg/kg.
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PMID:Synthesis and biological activity of ketomethylene pseudopeptide analogues as thrombin inhibitors. 152 87

Plasminogen activator and urokinase are often used as biological markers of cell activation. However, the methods currently used are cumbersome, make no discrimination between tissue-type plasminogen activator and urokinase, and do not allow expression of the results of the overall reaction in International Units. The one-step method described in this paper lacks these drawbacks. Moreover, we propose use of H-D-Val-Phe-Lys-4-nitroanilide as substrate which has a lower Km than the standard H-D-Val-Leu-Lys-4-nitroanilide which is commercially available. Low concentrations of sodium dodecyl sulfate in the reaction mixture dramatically and preferentially accelerate the reaction catalyzed by tissue-type plasminogen activators. Identical results are obtained under kinetic or fixed-time assay conditions using either a photometer or 96-well plate reader. The corresponding formulae are provided.
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PMID:Spectrophotometric method to quantify and discriminate urokinase and tissue-type plasminogen activators. 153 70

In this report, we investigated the expression and activation of proteolytic enzymes by mouse T-lymphoma cell lines of differing metastatic potential. In contrast to the low metastatic Eb line, the metastatic variants ESb and ESb-MP secreted urokinase-type plasminogen activator (u-PA), which was present in the culture supernatant predominantly in the active form (ESb, 96%; ESb-MP, 80%). All three T-lymphoma variants expressed a mainly cell surface-associated proteinase, which proved to be immunologically and enzymatically related to the murine T-cell-associated serine proteinase-1 (MTSP-1). Intact lymphoma cells were able to activate the recombinant human proenzyme of u-PA (pro-u-PA) by a plasmin-independent mechanism, because plasmin contamination of the cells was not detectable. When ESb-MP cells were cultured in the presence of inhibitors of MTSP-1, such as antithrombin III, Pro-Phe-Arg-chloromethylketone, or aprotinin, the ratio of endogenously activated murine u-PA to inactive pro-u-PA in conditioned medium was significantly reduced (from 80% to 15%). The most potent inhibitor, antithrombin, did not inhibit plasmin-catalyzed pro-u-PA activation. These results suggest a novel autocrine mechanism of plasmin-independent pro-u-PA activation for metastatic T lymphomas by the production of an MTSP-1-related proteinase. The ability to initiate the proteolytic cascade of plasminogen activation in the absence of plasmin might contribute to the metastatic behavior of these cells observed in vivo.
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PMID:A T-cell-related proteinase expressed by T-lymphoma cells activates their endogenous pro-urokinase. 156 36


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