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)

Protein C inhibitor (PCI), a glycosaminoglycan (GAG) dependent serine protease inhibitor, inhibits its target proteases by forming SDS-stable 1:1 complexes. GAGs alter target enzyme specificity of PCI in such a way that e.g. urokinase (uPA) is the preferred target enzyme in the presence of GAGs while in their absence preferentially tissue kallikrein (TK) complexes are formed. The effect of the GAG-binding adhesive glycoprotein vitronectin (Vn) on the GAG-stimulated inhibition of uPA by PCI was studied using an amidolytic assay. In the presence of heparin, Vn protected uPA from inhibition by PCI in a dose-dependent manner with respect to both, Vn- and heparin-concentration. Vn also was active when heparin was replaced by low-molecular weight heparin or heparan sulfate, respectively. In the absence of GAGs, Vn had no effect on the inhibition of uPA by PCI. In a similar system, Vn was far less effective in modifying the inhibitory function of heparin on the inhibition of TK by PCI. When equimolar concentrations of radiolabelled uPA and TK were incubated with PCI in the presence of heparin, only complexes of PCI with uPA were detectable. Addition of Vn reduced this complex formation, whereas, in contrast, complexes of PCI and TK appeared. These results indicate that Vn modulates both, the activity and specificity of PCI and suggest different structural heparin-requirements for the PCI/uPA versus PCI/TK interaction.
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PMID:Vitronectin modulates glycosaminoglycan dependent reactions of protein C inhibitor. 128 93

Proteinase species secreted by 10 human gastric carcinoma cell lines were analyzed by gelatin zymography and immunoblotting. These cell lines were classified into the following three groups with respect to proteinase secretion: cell lines secreting mainly gelatinases A and/or B; those secreting multiple types of serine proteinases; and those scarcely secreting these enzymes. Two cell lines of the second group, STKM-1 and MKN28, hardly secreted metalloproteinases but secreted the following four types of serine proteinases: (a) two trypsin-like enzymes (M(r) 26,000 and 24,000 in proenzyme forms); (b) a tissue kallikrein-like enzyme (M(r) 150,000 in a complex form); (c) a plasmin-like enzyme (M(r) 70,000); and (d) a plasminogen activator (urokinase-type, M(r) 57,000, from STKM-1 and tissue-type, M(r) 70,000, from MKN28). The M(r) 70,000 plasmin-like enzyme was also detected at lower levels in the conditioned media of four other cell lines (MKN1, MKN45, NUGC-3, and KATO III). The M(r) 24,000 proenzyme of the trypsin-like enzyme was purified from the serum-free conditioned medium of STKM-1. The proenzyme was activated by enterokinase treatment or autolytically by incubation at neutral pH, decreasing its apparent molecular weight from 24,000 to 23,000 on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The activated enzyme extensively degraded fibronectin, laminin, and gelatins and to lesser extents type I, III, IV, and V collagens at 30 degrees C. These results suggest that the matrix serine proteinases may play a major role in the matrix degradation by some kinds of human cancer cells.
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PMID:Multiple secretion of matrix serine proteinases by human gastric carcinoma cell lines. 138 87

It was shown that 7-amino-4-methylcoumarin (MC-amine), resulted from the enzymatic hydrolysis of 4-methylcoumaryl-7-amide (MC-amide) peptide substrates, may be estimated not only fluorometrically but also photometrically. A photometric method for estimating activity of tissue kallikrein (EC 3.4.21.35) and urokinase (EC 3.4.21.31) is suggested using Z-Phe-Arg-NHMC and Z-Gly-Gly-Arg-NHMC, respectively, as substrates. Kinetic parameters of the enzymatic hydrolysis, as obtained by photometric and fluorometric detection of the MC-amine formed, were in good agreement. The differential coefficient of molar extinction of the substrates and MC-amine at 360 nm was found to be 10,800 M-1 cm-1.
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PMID:[Photometric method of determination of the amidase activity of proteinases using 4-methylcoumaryl-7-amide substrates]. 240 Apr 5

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

Monoclonal antibodies to purified human urinary kallikrein have been developed. Selection of antibody producing clones was based on 125I-kallikrein binding activity of hybridoma media in both radioimmunoassay and enzyme-linked immunosorbent assay. Three clones (2 IgG1, 1 IgG2b) were subcloned, characterized, and compared with the polyclonal antiserum generated in rabbits immunized with the purified kallikrein. With radioimmunoassay, mouse ascitic fluids or rabbit antisera dilutions showing 50% binding to 125I-kallikrein were 1:1.2 X 10(6) (E7A9), 1:1.2 X 10(5) (H6A6), 1:8.0 X 10(4) (E12H1), and 1:1.4 X 10(6) (the rabbit antisera). With enzyme-linked immunosorbent assay, mouse ascitic fluids from clones E7A9 and H6A6 showed half-maximal absorbance at dilutions of 1:2.1 X 10(5) and 1:1.0 X 10(5) respectively, and the polyclonal antiserum showed half-maximal absorbance at a dilution of 1:2.0 X 10(4). These monoclonal antibodies showed no cross-reactivity with rat tissue kallikrein, rat urinary plasminogen activator, or dog pancreatic kallikrein, while the polyclonal antiserum showed some cross-reactivity. The binding of monoclonal or polyclonal antibodies to 125I-human urinary kallikrein was not affected by human plasma kallikrein, thrombin, or urokinase in a competitive radioimmunoassay. By using purified human urinary kallikrein immobilized to agarose, antibodies produced by clones E7A9 and H6A6 and in the rabbit antisera were purified to homogeneity. Each of these affinity-purified antibodies inhibited the esterase activity, and two of the three inhibited the kininogenase activity, of human urinary kallikrein. A sandwich immunosorbent assay was developed to measure this kallikrein using monoclonal antibody from the clone E7A9 in conjunction with the polyclonal antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of monoclonal and polyclonal antibodies to human tissue kallikrein. 385 80

Rat urinary esterase A, a plasminogen activator with kininogenase activity, was recently purified and characterized (J. Chao (1983) J. Biol. Chem. 258, 4434-4439). A sensitive radioimmunoassay for esterase A has been developed. This assay uses a rabbit antiserum in a final dilution of 1:160 000 and the purified enzyme was labelled with 125I using a lactoperoxidase method. It detects 80 pg of immunoreactive material per tube. This antiserum has some cross-reactivity with rat urinary kallikrein (approximately 5%) but a previously characterized tissue kallikrein antiserum has negligible cross-reactivity with the urinary esterase A in the assays. Therefore, kallikrein levels are measured simultaneously in all samples to obtain accurate levels of immunoreactive esterase A. Dilutions of urine or tissue homogenates showed complete parallelism with esterase A standard curves. No cross-reactivity with dog, human or monkey urine was seen. The recovery of esterase A from rat urine was 99.7 +/- 3.5%. Intra- and between-assay errors were 6.5 and 11.2%, respectively. Immunoreactive esterase A was measured and compared with kallikrein levels in rat urine, kidney, pancreas, submandibular gland, descending colon and ileum. The urinary esterase A excretion rate was reduced significantly in rats on a high sodium, compared with a low sodium diet, but not significantly increased above control by the latter. Nonetheless, a significant correlation between urinary kallikrein and esterase A excretion rate was present. This radioimmunoassay can now be used to measure esterase A levels in urine and tissue as questions have arisen about its regulation and functional significance.
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PMID:Measurement of the rat urinary plasminogen activator (esterase A) by direct radioimmunoassay in urine and tissue. 656 99

Direct muscle injury was induced in rats in order to evaluate alterations in the balance of serine proteases and inhibitors (serpins) as a response to tissue damage. It was previously found that certain proteases, specifically urokinase-like plasminogen activator (uPA) and others, required activation in order to effect regeneration. We hypothesized that the magnitude and temporal sequence of serpin activation would follow, pari passu, activation of their cognate proteases. In addition to uPA, tissue PA (tPA) and tissue kallikrein were the proteases studied. The serpins we analyzed were protease nexin I (PNI), PA inhibitor 1 (PAI-1, and the kallikrein-binding protein (KBP). uPA nearly doubled 48 h after injury, while there was no change in amidolytic activity after addition of fibrin monomer as an estimation of tPA activity. Tissue kallikrein activity, barely detectable in normal muscle, slowly increased, nearly tripling at 7 days after injury. Greater magnitude and more rapid changes in muscle serpins occurred over the same post-injury time course. By 24 h PNI increased threefold, while PAI-1 increased more slowly, reaching double the control values by 5 days after injury. Surprisingly, KBP, the serpin-class inhibitor of tissue kallikrein, had the most robust response, increasing tenfold over control 48 h after crush injury of muscle. These results further implicate the serpin:protease balance in tissue injury. Participation of complex receptors, such as the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP), various growth factors, cytokines, and other molecules, in regulating this balance is implicated by these data.
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PMID:Activation of serpins and their cognate proteases in muscle after crush injury. 813 78

Human seminal plasma trypsin-like proteinase inhibitor (HSTPI) was separated and examined by trypsin Cellulofine affinity adsorption and Cellulofine GCL-300 gel filtration and its inhibitory action toward some arginine amidases obtained from the urine, semen, and blood of humans. HSTPI showed strong inhibitory action toward two types of human seminal plasma basic arginine amidases (BHSAA-L and -A), human seminal plasma acidic arginine amidase with affinity to lima bean trypsin inhibitor (LBTI) column (AHSAA-L), and human acrosin and thrombin. Conversely, no or little inhibition was observed toward human urinary arginine amidase-2, human high molecular weight urokinase, or human seminal plasma acidic arginine amidase with affinity to aprotinin column (AHSAA-A, tissue kallikrein). Measurement of Ki values of BHSAA-L with affinity to LBTI column toward HSTPI and LBTI revealed that the arginine amidase had a stronger affinity for LBTI than that for HSTPI. This indicates that it is the difference in Ki values that allows BHSAA-L to be separated by the LBTI affinity adsorption method from human seminal plasma containing a large amount of HSTPI.
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PMID:Human seminal plasma proteinase inhibitor: action toward some trypsin-like arginine amidases from humans. 837 82

Tryptase is a serine protease secreted by mast cells that is able to activate other cells. In the present studies we have tested whether these responses could be mediated by thrombin receptors or PAR-2, two G-protein-coupled receptors that are activated by proteolysis. When added to a peptide corresponding to the N terminus of PAR-2, tryptase cleaved the peptide at the activating site, but at higher concentrations it also cleaved downstream, as did trypsin, a known activator of PAR-2. Thrombin, factor Xa, plasmin, urokinase, plasma kallikrein, and tissue kallikrein had no effect. Tryptase also cleaved the analogous thrombin receptor peptide at the activating site but less efficiently. When added to COS-1 cells expressing either receptor, tryptase stimulated phosphoinositide hydrolysis. With PAR-2, this response was half-maximal at 1 nM tryptase and could be inhibited by the tryptase inhibitor, APC366, or by antibodies to tryptase and PAR-2. When added to human endothelial cells, which normally express PAR-2 and thrombin receptors, or keratinocytes, which express only PAR-2, tryptase caused an increase in cytosolic Ca2+. However, when added to platelets or CHRF-288 cells, which express thrombin receptors but not PAR-2, tryptase caused neither aggregation nor increased Ca2+. These results show that 1) tryptase has the potential to activate both PAR-2 and thrombin receptors; 2) for PAR-2, this potential is realized, although cleavage at secondary sites may limit activation, particularly at higher tryptase concentrations; and 3) in contrast, although tryptase clearly activates thrombin receptors in COS-1 cells, it does not appear to cleave endogenous thrombin receptors in platelets or CHRF-288 cells. These distinctions correlate with the observed differences in the rate of cleavage of the PAR-2 and thrombin receptor peptides by tryptase. Tryptase is the first protease other than trypsin that has been shown to activate human PAR-2. Its presence within mast cell granules places it in tissues where PAR-2 is expressed but trypsin is unlikely to reach.
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PMID:Interactions of mast cell tryptase with thrombin receptors and PAR-2. 902 Jan 12

The non-specific serine-protease inhibitor protein-C inhibitor (PCI) inactivates its target enzymes by forming stable 1:1 complexes. Heparin stimulates most PCI/protease reactions, but interferes with the inhibition of tissue kallikrein by PCI by a hitherto unknown mechanism. In this study we analyzed the inhibitory effect of heparin on the tissue-kallikrein-PCI interaction. Free PCI and tissue-kallikrein x PCI complexes but not free tissue kallikrein bound to heparin-Sepharose, implying that the inhibitory effect of heparin cannot be caused by a tissue-kallikrein-heparin interaction. Heparin did not dissociate tissue-kallikrein x PCI complexes, making it unlikely that in the presence of heparin PCI becomes a substrate for, rather than an inhibitor of, tissue kallikrein. However, heparin-bound PCI, which was able to form complexes with 125I-urokinase, did not form complexes with 125I-tissue-kallikrein. This suggests that the inhibitory effect of heparin is either based on the neutralization of positive charges in the PCI molecule, which might be required for the interaction of PCI with the acidic protease tissue kallikrein, or on a change in reactivity of PCI upon heparin binding, making heparin-bound PCI no longer a tissue-kallikrein inhibitor. Neutralization of basic amino acids in the PCI molecule by glutamic acid, which prevented in a dose-dependent way the inhibitory effect of heparin, did not have any effect on the tissue-kallikrein-PCI interaction. Therefore, direct involvement of basic amino acid residues present in the heparin-binding site of PCI in the tissue-kallikrein-PCI interaction can be excluded. Heparin binding might rather cause a change in reactivity of PCI (e.g. by inducing a conformational change or by steric interference), thereby preventing its interaction with tissue kallikrein.
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PMID:Heparin binding of protein-C inhibitor--analysis of the effect of heparin on the interaction of protein-C inhibitor with tissue kallikrein. 934 5


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