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)

S-S cross-linking enzyme, skin sulfhydryl oxidase (SSO), catalyzes the formation of disulfide bonds from sulfhydryl groups in skin. The activity of SSO was detected in differing amounts in each of the four layers--stratum corneum, stratum granulosum, stratum spinosum with basal cell layer, and dermis--of cow snout skin, with the highest specific activity being recorded in the stratum granulosum. SSO was stimulated to 130-150% of its initial activity by treatment with 1 mg/ml trypsin, chymotrypsin, or urokinase, but was not affected by plasmin or cathepsin D. These findings suggest that SSO may be activated by some kinds of serine proteases during the keratinocyte autolysis process in the stratum granulosum. SSO showed the highest activity with the addition of 5 microM of Cu2+. The atomic absorptive analysis of purified SSO showed 0.5 atoms of Cu in one molecule of SSO. From these findings, it was determined that Cu2+ was essential for the activity of SSO. The molar ratio of the disappearance of DTT, consumption of O2, and production of H2O2 during the enzyme reaction was 1:1.05:0.89. From these findings, the reactions catalyzed by SSO is suggested to be represented by the following equation: (table; see text).
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PMID:[Localization in skin, activation and reaction mechanisms of skin sulfhydryl oxidase]. 258 80

The inhibitory effect of the clinically used p-carbethoxyphenyl ester of epsilon-guanidino-caproic acid methanesulphonate (epsilon-GCA-CEP) on the catalytic properties of human LYS77-plasmin (EC 3.4.21.7), bovine factor Xa (EC 3.4.21.6), bovine alpha-thrombin (EC 3.4.21.5), ancrod (EC 3.4.21.28), crotalase (EC 3.4.21.30), bovine beta-trypsin (EC 3.4.21.4), porcine pancreatic beta-kallikrein-B (EC 3.4.21.35), human urinary kallikrein (EC 3.4.21.35) and the Mr 54,000 species of human urokinase (EC 3.4.21.31) was investigated (between pH 2.0 and 8.5, I = 0.1 M; T = 21 +/- 0.5 degrees C), and analyzed in parallel with that of the homologous derivative p-carbethoxyphenyl epsilon-amino-caproate hydro chloride (epsilon-ACA-CEP). On lowering the pH from 5.5 to 3.0, values of the apparent dissociation inhibition constant (Ki) for epsilon-GCA-CEP and epsilon-ACA-CEP interaction with the serine proteinases considered increase, reflecting the acidic pK-shift upon inhibitor binding of a single ionizing group. Over the whole pH range explored, (i) epsilon-GCA-CEP interacts with bovine factor Xa and bovine alpha-thrombin with an higher affinity than that observed for epsilon-ACA-CEP binding; (ii) both inhibitors associate to bovine beta-trypsin with the same affinity; and (iii) epsilon-ACA-CEP inhibits human Lys77-plasmin and the Mr 54,000 species of human urokinase with an higher affinity than that reported for epsilon-GCA-CEP association, thus reflecting the known enzyme primary specificity properties. However, the affinity of epsilon-ACA-CEP for ancrod, crotalase, porcine pancreatic beta-kallikrein-B and human urinary kallikrein, all of which preferably bind arginyl rather than lysyl side chains at the primary position of substrates and/or inhibitors, is paradoxically higher than that displayed by epsilon-GCA-CEP. By considering the amino acid sequences, the X-ray three-dimensional structures and/or the computer-generated molecular models of serine proteinase: inhibitor adducts, the observed binding behaviour of epsilon-GCA-CEP and epsilon-ACA-CEP to the enzymes considered has been related to the inferred stereochemistry of proteinase: inhibitor contact region(s).
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PMID:Inhibition of serine proteinases by p-carbethoxyphenyl esters of epsilon-guanidino- and epsilon-amino caproic acid: thermodynamic and molecular modeling study. 272 72

A method for the determination of the catalytic parameters Ks, k+2 and k+3 describing trypsin-like serine proteinase action has been developed from the quantitative analysis of the kinetics of hydrolysis of two specific chromogenic substrates, N-alpha-carbobenzoxy-L-arginine p-nitrophenyl ester and N-alpha-carbobenzoxy-L-lysine p-nitrophenyl ester, catalyzed by porcine pancreatic betta-kallikrein B, bovine betta-trypsin and human urokinase (Mr 54,000 species), under conditions where the concentration of the substrate exceeds that of the enzyme. Value of Ks, k+2 and k+3 have been estimated from the effect of substrate concentration on the apparent first-order rate constant of the time-course of the burst phase of p-nitrophenol release preceding the steady-state reaction.
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PMID:Trypsin-like serine proteinase action: determination of the catalytic parameters KS, k+2 and k/3 under conditions where the substrate exceeds the enzyme concentration. 279 64

Previous studies have shown that neuroblastoma cells and several types of primary neuronal cells in culture rapidly extend neurites when switched from serum-containing to serum-free medium. The present studies on cloned neuroblastoma cells show that thrombin blocked this spontaneous differentiation at 2 nM with a half-maximal potency of 50 pM. This required the catalytic activity of thrombin and was reversed upon thrombin removal. Thrombin also caused cells in serum-free medium to retract their neurites at equally low concentrations. Two other serine proteases, urokinase and plasmin, did not block or reverse neurite extension even at 100-fold higher concentrations. A specific assay for thrombin indicated that thrombin detected in serum-containing medium from neuroblastoma cultures was derived from serum and that it was likely responsible for much of the known capacity of serum to maintain neuroblastoma cells in a nondifferentiated state. This was supported by the finding that heparin addition reduced the thrombin concentration in serum-containing medium and stimulated neurite outgrowth from neuroblastoma cells in serum-containing medium. Studies on the ability of thrombin to modulate neurite outgrowth by other agents showed that it blocked and reversed the neurite outgrowth activity of two thrombin inhibitors: protease nexin-1 (which is identical to glial-derived neurite-promoting factor) and hirudin. Thrombin, however, did not block the neurite-promoting activity of dibutyryl cAMP or prostaglandin E1. These results suggest a specific role for thrombin in control of neurite outgrowth.
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PMID:Thrombin modulates and reverses neuroblastoma neurite outgrowth. 283 73

The kinetics of the activation of plasminogen by recombinant pro-urokinase obtained by expression of human urokinase cDNA in Escherichia coli was studied. The conversion of pro-urokinase (U) and plasminogen (P) to urokinase (u) and plasmin (p) is represented by a sequence of three reactions which each obey Michaelis-Menten kinetics, i.e. (Formula: see text). In this model, pro-urokinase formally behaves as an enzyme in Reaction I and as a substrate in reaction II. The experimentally measured overall rates of formation of urokinase and plasmin are in good agreement with those calculated from the kinetic parameters and the initial concentrations of pro-urokinase and plasminogen, confirming the validity of the model. It appears that recombinant pro-urokinase is an equally potent activator of plasminogen (k2/Km = 0.05 microM-1 s-1), as in urokinase (k"2/K"m = 0.02 microM-1 s-1). This is due to the fact that the proenzyme, which is virtually inactive toward low Mr substrates for urokinase, forms an intermediate of the Michaelis-Menten type with plasminogen, with a much higher affinity than that of the active enzyme with its substrate. This is an exceptional phenomenon among the serine proteases.
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PMID:Activation of plasminogen by pro-urokinase. II. Kinetics. 293 29

The turnover of basement membrane macromolecules in injured skeletal muscle has not been studied in contrast to other biologic systems undergoing remodeling. Plasminogen activators and other neutral proteases that are able to degrade these basement membrane macromolecules are secreted by cultured muscle cells. We sought to determine if locally released plasminogen activators could act on basement membrane components. Such degradation might be implicated in the disadhesion of nerve from muscle after motor nerve denervation. To test this hypothesis, we first undertook a study of the sensitivity of muscle extracellular matrix antigens following in vitro exposure to various proteases on frozen muscle sections. Fibronectin was found to be most sensitive, followed by type IV collagen and laminin. Of serine proteases, trypsin was the most active but was not selective, digesting matrix and sarcoplasmic components alike in less than 30 min. Purified urokinase was inactive unless plasminogen (also inactive alone) was previously added to tissue sections, at which time only matrix antigens were digested. Little if any observable degradation of sarcoplasmic proteins took place under these conditions. Using a highly sensitive and selective assay, we found that plasminogen activators were present in muscle tissue and increased 8- to 10-fold after 10 days of denervation. Using an extract of denervated muscle in the presence of plasminogen, we observed degradation of matrix antigens. No degradation was observed with control muscle extract. We next evaluated the degradation of these antigens in denervated muscle during a temporal study. The results, analyzed by quantitative image analysis, indicates that with increasing time after denervation a marked decrease of fibronectin and type IV collagen, followed by laminin occurred but, again, only in the present of plasminogen. These results indicate a selective sensitivity of basement membrane antigens of muscle and a role for plasminogen activators in the degradation of these adhesive basement membranes macromolecules after denervation.
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PMID:Degradation of muscle basement membrane zone by locally generated plasmin. 294 9

We have compared the plasminogen activating capacity of one- and two-chain urokinase-type plasminogen activator (u-PA). In a 125I-plasminogen conversion assay in the presence of high amounts of a plasmin inhibitor, one-chain u-PA pretreated with diisopropyl fluorophosphate had no detectable activity, the detection limit corresponding to the activity of a 400-fold lower amount of two-chain u-PA. In coupled assays in which generated plasmin was measured with a synthetic substrate, activity was clearly observed with the one-chain preparation, but the initial rate of plasminogen activation was lower than that of a 250-fold smaller concentration of two-chain u-PA. The coupled assays for one-chain u-PA are self-activating because plasmin catalyzes conversion of one- to two-chain u-PA, and it is not possible to decide whether the low activity of one-chain u-PA observed with this type of assay is intrinsic or due to contaminations. On the basis of these findings and a discussion of previous studies, it is concluded that one-chain u-PA has a variety of properties similar to the one-chain proenzyme forms of other serine proteases and that it should, therefore, be considered as a genuine proenzyme form of u-PA.
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PMID:One-chain urokinase-type plasminogen activator from human sarcoma cells is a proenzyme with little or no intrinsic activity. 296 91

Lp(a) represents a genetically transmitted class of plasma LDL having apo B-100 linked by a disulfide bridge to a glycoprotein, apo(a). Lp(a) is heterogeneous in size and density. Apo(a) is also heterogeneous in size (molecular weight between approximately 300,000 and 700,000) due probably to the polymorphism of both polypeptide and carbohydrate chains. Recent studies have shown that apo(a) has a striking amino acid sequence homology with plasminogen, a serine protease zymogen that following activation to plasmin enters the fibrinolytic system. Apo(a) is severalfold larger than plasminogen (molecular weight approximately 90,000) and also differs from it because it fails to be activated to plasmin. This is due to the fact that arginine is replaced by serine at the site of cleavage by streptokinase, urokinase, or tissue plasminogen activator. A single gene locus appears to control the Lp(a) polymorphism as well as the concentration of the Lp(a) phenotypes in the plasma. Patients with high plasma levels of Lp(a) have been shown to have an increased incidence of cardiovascular disease but a causal relationship has not been firmly established. The information that is being rapidly acquired on the structure of Lp(a) should facilitate the understanding of the molecular basis of the polymorphism of this genetic variant and of the role that the various Lp(a) phenotypes play in atherosclerosis and thrombosis. The potential physiologic role of Lp(a) remains open to inquiry.
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PMID:Lipoprotein(a): a genetically determined lipoprotein containing a glycoprotein of the plasminogen family. 297 66

Affinity-purified antibody against human factor XII (Hageman factor) has been radiolabeled with 125I and employed as a probe to screen a human liver cDNA expression library prepared in lambda gt11. Approximately 3.5 X 10(6) recombinant phages were screened for factor XII, and two positive clones were identified and plaque purified. The largest cDNA coding for factor XII was 1571 base pairs in length and coded for amino acid residues 127-596 in the mature protein, a termination codon of TGA, a 3' noncoding sequence of 147 nucleotides, and a poly(A) tail of 11 nucleotides. The second clone contained an insert of 1334 base pairs and coded for amino acid residues 200-596. The amino acid sequence predicted by the cDNAs was in excellent agreement with that previously determined by amino acid sequence analysis. The amino acid and DNA sequences in human factor XII showed considerable homology with the corresponding domains in other serine proteases, including prothrombin, plasminogen, tissue plasminogen activator, and urokinase.
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PMID:Characterization of a cDNA coding for human factor XII (Hageman factor). 301 Oct 63

Fibroblasts as well as several other cell types, secrete a number of protease inhibitors into their culture media. Among these inhibitors are the protease nexins, a class of proteins which covalently bind serine proteases, thereby inactivating their specific targets. Protease nexin-I, first discovered in human foreskin fibroblasts, binds thrombin, plasmin, and urokinase with high affinity, forming covalently linked complexes. Human fibroblasts bind complexes of protease nexin-I and its target protease via a cell-surface, high-affinity receptor. We have analyzed a number of characteristics of this receptor, and found them to be typical of class II receptors in general. At 4 degrees C binding of PN-I:protease complexes was competed by heparin. In addition, binding was independent of the particular protease bound to the PN-I; purified complexes of PN-I with thrombin or urokinase competed equipotently for [125]I-thrombin:PN-I binding. As the pH of the binding buffer was lowered, binding to cells increased. A twofold increase in binding was attained by lowering the pH from 7.5 to 4.5. This phenomenon was not due to irreversible, pH-induced changes to either the cell surface or the labeled complexes. At 37 degrees C, the removal of labeled complexes from culture medium was rapid; approximately 80% was removed by 4 hours under given conditions. The internalization of complexes was also very rapid, with an estimated ke (endocytic rate constant) of 1.0 min-1. At neutral pH, fibroblasts bind complexes in a saturable manner. Scatchard analysis yields a receptor number of 250,000 per cell and a Kd of 1 nM.
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PMID:Characterization of the receptor for protease nexin-I:protease complexes on human fibroblasts. 303 24


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