Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have isolated heparan sulfate proteoglycans (HSPGs) from cloned rat microvascular endothelial cells using a combination of ion-exchange chromatography, affinity fractionation with antithrombin III (AT III), and gel filtration in denaturing solvents. The anticoagulantly active heparan sulfate proteoglycans (HSPGact) which bind tightly to AT III bear mainly anticoagulantly active heparan sulfate (HSact) whereas the anticoagulantly inactive heparan sulfate proteoglycans (HSPGinact) possess mainly anticoagulantly inactive heparan sulfate (HSinact). HSact and HSinact were also isolated by a combination of ion-exchange chromatography, treatment with protease and chondroitin ABC lyase, and affinity fractionation with AT III. HSact and HSinact have molecular sizes of about 25-30 kDa with the same overall composition of monosaccharides except that HSact exhibits about nine glucuronsyl 3-O-sulfated glucosamines/chain whereas HSinact possesses about three glucuronsyl 3-O-sulfated glucosamines/chain. Direct isolation of the AT III-binding site of HSact by exposing carbohydrate chains to Flavobacterium heparitinase in the presence of protease inhibitor revealed only a single interaction site which contained two to three glucuronsyl 3-O-sulfated glucosamine residues. The core proteins of HSPGact and HSPGinact were isolated by treatment with Flavobacterium heparitinase and purification by ion-exchange chromatography. The molecular sizes of the core proteins were established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their primary structures were examined by cleavage with trypsin or endopeptidase Glu-C as well as separation of peptides by reverse-phase high performance liquid chromatography. The results showed that both sets of core proteins exhibited three major components with molecular sizes of 50, 30, and 25 kDa, respectively. The 25-kDa species appears to be a proteolytic degradation product of the 30-kDa species. The peptide mapping revealed that HSPGact and HSPGinact possess extremely similar core proteins.
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PMID:Isolation and characterization of heparan sulfate proteoglycans produced by cloned rat microvascular endothelial cells. 153 64

Formation of the covalently stabilized alpha 1-antitrypsin (alpha 1-AT)-neutrophil elastase complex, the archetype of serpin-enzyme complexes, results in a structurally rearranged alpha 1-AT molecule that possesses chemo-attractant activities, mediates an increase in synthesis of alpha 1-AT by mononuclear phagocytes and hepatocytes, and is more rapidly cleared from the circulation than is the native alpha 1-AT molecule. We have recently identified an abundant, high affinity cell surface receptor on human hepatoma HepG2 cells and human monocytes that binds alpha 1-AT-elastase complexes, mediates endocytosis and lysosomal degradation of alpha 1-AT-elastase complexes, and induces an increase in synthesis of alpha 1-AT. We have referred to this receptor as the serpin-enzyme complex, or SEC, receptor because it also recognizes complexes of serpins antithrombin III, alpha 1-antichymotrypsin, and C1 inhibitor with their cognate enzymes. In the current study, we show that a pentapeptide domain in the carboxyl terminal fragment of alpha 1-AT (amino acids 370-374, FVFLM) is sufficient for binding to the SEC receptor. A synthetic analog of this pentapeptide (peptide 105C, FVYLI) blocks binding and internalization of alpha 1-AT-125I-trypsin complexes by HepG2 cells. 125I-Peptide 105C binds specifically and saturably to HepG2 cells, and its binding is blocked by alpha 1-AT-trypsin or alpha 1-AT-elastase complexes. Alterations of this sequence introduced into synthetic peptides (mutations, deletions, or scrambling) demonstrate that binding of the pentapeptide domain is sequence-specific. Comparisons with the sequences of other serpins in the corresponding region indicate that this pentapeptide neodomain is highly conserved.
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PMID:The SEC receptor recognizes a pentapeptide neodomain of alpha 1-antitrypsin-protease complexes. 164 29

Adenosine 3',5'-cyclic monophosphate (cAMP) elevation in cultured rat mesangial cells causes urokinase-dependent adhesion loss, stress-fiber fragmentation, and shape change. Thrombin cleaves single-chain urokinase (scu-PA), causing its inactivation, but not two-chain u-PA [tcu-plasminogen activator (PA)] or tissue-type PA. We tested the ability of thrombin to inhibit the effects of cAMP elevation in mesangial cells and inactivate cell-associated scu-PA. In an assay of trypsin-sensitive adhesion, 65.9% of control cells and 5.5% of cells treated with isoproterenol + methylisobutylxanthine (IM) remained adherent. In the presence of 0.01, 0.1, 1.0, and 10.0 unit/ml thrombin, 20.9, 46.6, 50.4, and 53.3%, respectively, of IM-treated cells remained attached. Thrombin also inhibited stress-fiber fragmentation and shape change. The effects of thrombin were blocked by hirudin or antithrombin III plus heparin. Direct zymography in gels containing gelatin and plasminogen revealed loss of a closely spaced pair of PA bands with thrombin treatment (1.0 unit/ml). Hirudin blocked the loss. alpha-Thrombin inactivated by diisopropyl fluorophosphate neither inhibited shape change nor caused loss of the PA bands; however, gamma-thrombin was nearly as active as native alpha-thrombin in both regards. Pretreatment of the cells with as little as 1.0 unit/ml thrombin for 1.0 min caused marked inhibition of shape change and near total loss of the slower migrating u-PA band (of the doublet). The faster migrating band was inhibited less. The results indicate that the slower migrating band represents scu-PA; the nature of the faster migrating band is less certain. Thrombin reversed the adhesion loss and shape change caused by 8-(4-chlorophenylthio)-cAMP and MIX. Thus physiological concentrations of thrombin rapidly inactivate mesangial cell scu-PA and inhibit and reverse cAMP-stimulated adhesion loss and shape change.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of mesangial cell adhesion and shape by thrombin. 165 8

The interaction between Porphyromonas gingivalis culture supernatant and human serum was examined. Hydrolysis of the major serum proteins was thiol-dependent and correlated with the trypsin-like activity of the sample. Transferrin and IgG light chains were less susceptible to degradation than albumin and IgG heavy chains and partially degraded IgG retained antigen-binding capability. Serum inhibited the trypsin-like activity in a fluorimetric assay. The inhibition was shown to be independent of the level of IgG antibody reactive with whole cells of P. gingivalis. Purified preparations of antithrombin III, a serine protease inhibitor, but not alpha 1-antitrypsin nor alpha 2-macroglobulin inhibited the trypsin-like activity in the fluorometric assay.
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PMID:Degradation of plasma proteins by the trypsin-like enzyme of Porphyromonas gingivalis and inhibition of protease activity by a serine protease inhibitor of human plasma. 166 33

Tryptase from human mast cells has been shown (in vitro) to catalyze the destruction of fibrinogen and high-molecular-weight kininogen as well as the activation of C3a and collagenase. Although large amounts of tryptase are released in tissues by degranulating mast cells and levels as high as 1000 ng/ml have been measured in the circulation following systemic anaphylaxis, no specific physiologic inhibitor has yet been found for the protease. The current work tests several more inhibitors for their effects on tryptase and examines any effect of tryptase on these inhibitors. First, antileukoprotease and low-molecular-weight elastase inhibitor from human lung and hirudin and antithrombin III had no effect on tryptase activity in vitro. Second, the possibility that tryptase, being insensitive to the effects of inhibitors, might instead destroy them was also considered. Tryptase failed to cleave and inactivate antileukoprotease, low-molecular-weight elastase inhibitor, alpha 1 protease inhibitor, alpha 2 macroglobulin, and antithrombin III. Third, based on the knowledge that tryptase stability is regulated by its interaction with heparin, antithrombin III was used as a model heparin-binding protein to demonstrate that a protein competitor for heparin-binding sites, presumably by displacement of tryptase, destabilizes this enzyme. Conversely, tryptase, in excess, blocked the binding of antithrombin III to heparin, thereby attenuating the heparin-mediated inhibition of thrombin by antithrombin III.
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PMID:Interactions of human mast cell tryptase with biological protease inhibitors. 168 95

Interaction of vitronectin with glia-derived nexin (GDN), thrombin, and the complex GDN-thrombin was demonstrated in direct binding assays that indicated the formation of binary and ternary complexes. The concentration of vitronectin necessary to obtain 50% saturation of the immobilized GDN-thrombin complex binding sites (EC50) was about 1 nM. Under similar experimental conditions, the EC50 of vitronectin for the immobilized antithrombin-III-thrombin complex was about fivefold higher. A tight complex was also formed between vitronectin and immobilized GDN (EC50 approximately 1.5 nM) but when vitronectin was immobilized, GDN displayed a reduced affinity for vitronectin (EC50 approximately 10 nM). These results suggest differences between the immobilized and free conformations of GDN and/or vitronectin. In contrast, vitronectin displayed negligible affinity for antithrombin III. Biotinylated GDN was used to characterize further the binding of GDN or the GDN-thrombin complex to vitronectin. The interaction of the biotinylated GDN-thrombin complex with immobilized vitronectin (EC50 approximately 2 nM) was completely blocked by nonbiotinylated complexes of thrombin with either GDN or antithrombin III, whereas free GDN, free thrombin and the GDN-trypsin complex were only weak competitors. Active-site-blocked urokinase and the complex GDN-urokinase also strongly competed for binding of the biotinylated GDN-thrombin complex to vitronectin. Binding of biotinylated GDN to immobilized vitronectin was specific, saturable and was competed with decreasing efficiency by the GDN-thrombin complex, free GDN and free antithrombin III. These interactions between the adhesive component vitronectin and the serine protease inhibitor GDN may relate to localized control of thrombin and/or urokinase action at certain extravascular sites. These results are discussed in terms of binding sites for vitronectin on GDN, thrombin, and the GDN-thrombin complex.
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PMID:Specific interaction of vitronectin with the cell-secreted protease inhibitor glia-derived nexin and its thrombin complex. 169 27

A plasma kallikrein inhibitor in guinea pig plasma (KIP) was purified to homogeneity. KIP is a single chain protein and the apparent molecular weight is estimated to be 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In amino acid composition, KIP is similar to human and mouse alpha 1-proteinase inhibitors and mouse contrapsin. KIP forms an equimolar complex with plasma kallikrein in a dose- and time-dependent fashion. The association rate constants for the inhibition of guinea pig plasma kallikrein by KIP, alpha 2-macroglobulin, C1-inactivator and antithrombin III were 2.5 +/- 0.3.10(4), 2.4 +/- 0.4.10(4), 6.6 +/- 0.5.10(4) and 9.1 +/- 0.6.10(2), respectively. Comparison of the association rate constants and the normal plasma concentrations of the four inhibitors demonstrates that KIP is ten-times as effective as alpha 2-MG and other two inhibitors are marginally effective in the inhibition of kallikrein. KIP inhibits trypsin and elastase rapidly, and thrombin and plasmin slowly, but is inactive for chymotrypsin and gland kallikrein. These results suggest that KIP is the major kallikrein inhibitor in guinea pig plasma and the proteinase inhibitory spectrum is unique to KIP in spite of the molecular similarity to alpha 1-proteinase inhibitor.
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PMID:The major plasma kallikrein inhibitor of guinea pig plasma. 173 48

Structural and functional properties of alpha-protease nexin I (alpha-PNI) expressed in Chinese hamster ovary cells were studied. All three cysteines were in the reduced form, showing that the potential disulfide bridge between residues Cys117 and Cys131 was not formed. Heparin association rate enhancements were from ka = 8.3 x 10(5) to 0.7-1.6 x 10(9) M-1 s-1 for the interaction of PNI with thrombin, from ka = 5.1 x 10(3) to 3.5 x 10(5) M-1 s-1 for interaction with Factor Xa, and from ka = 2.2 x 10(6) to 1.0 x 10(7) M-1 s-1 for interaction with trypsin; there was no rate enhancement of the plasmin interaction (ka = 1.0 x 10(5) M-1 s-1). The minimal heparin pentasaccharide had no effect on these interactions. Cleavage of the reactive center loop of PNI by three different proteases gave the typical stressed to relaxed change in thermal stability, but unlike with antithrombin III, there was no loss of heparin affinity. A similar difference from antithrombin was that PNI-thrombin complexes retained normal heparin affinity. These results are compatible with a role for protease nexin I as a cell-associated thrombin inhibitor that remains bound to the cell surface even after complexing with the protease, as compared with the role of antithrombin III as a circulating inhibitor of thrombin that becomes activated on binding to the microvasculature and is released on complex formation.
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PMID:Protease specificity and heparin binding and activation of recombinant protease nexin I. 193 53

The coagulation disturbance observed during severe acute pancreatitis before and after the infusion of a new synthetic low molecular weight protease inhibitor (Fut-175) was compared. The coagulo-fibrinolytic changes after acute pancreatitis was induced by the intraductal injection of an autologous bile and trypsin mixture showed decreased platelet counts, decreased plasma fibrinogen levels, prolonged partial prothrombin time and increased fibrinogen degradation products. In addition, markers of hypercoagulation showed increased fibrin-peptide A and decreased antithrombin III. The two markers of fibrinolysis showed increased B beta 15-42 immunoreactive peptide and decreased alpha 2 antiplasmin. After the infusion of Fut-175, the coagulo-fibrinolytic abnormalities, which were observed during severe acute pancreatitis without infusion of Fut-175, were improved. Furthermore, Fut-175 could suppress the rise in fibrino-peptide A and B beta 15-42 immunoreactive peptide and decrease in antithrombin III and alpha 2 antiplasmin. Thus, Fut-175 seems to be an effective inhibitor of protease-mediated hypercoagulation and fibrinolysis in severe acute pancreatitis.
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PMID:Effect of a synthetic protease inhibitor (Fut-175) on coagulation abnormalities during experimental acute pancreatitis in dogs. 227 34

We have recently reported that exogenous thrombin produced a dose- and endothelium-dependent coronary vasodilation in both intact open-chested dogs and in isolated dog coronary artery preparations. To determine whether the observed vasodilatory effect may be related to thrombin proteolytic enzymatic activity, effects of other proteases, such as trypsin, chymotrypsin, and pepsin, on the mechanical responses of isolated dog coronary arteries were studied. Among the four proteases evaluated, only thrombin (0.01-0.1 U/ml) and trypsin (0.03-0.67 U/ml) consistently produced a potent dose- and endothelium-dependent relaxation, that was reproducible with repeated testings. Addition of chymotrypsin (0.01-1.0 U/ml) produced only a minimal effect and was not reproducible, while addition of pepsin, as much as 10 U/ml, did not produce any effect. The specific soybean trypsin inhibitor and aprotinin, but not heparin and hirudin, competitively shifted the trypsin dose-response to the right, whereas heparin, hirudin, and antithrombin III proved to be more effective than trypsin inhibitors in inhibiting the thrombin-induced vasodilation. In all cases, the thrombin- and trypsin-induced vasodilation were equally sensitive to inhibition by the specific synthetic thrombin inhibitor, PPACK (D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone, 1-30 nM). PPACK, however, had no effect on the other endothelium-dependent coronary vasodilators, such as acetylcholine and adenosine triphosphate, in our isolated dog coronary artery preparations. Biochemical determinations of the amidolytic activity of thrombin, using Tosylglycyl-L-prolyl-L-arginine-p-nitroanilide as a chromogen, also indicated a similar PPACK and heparin-antithrombin III dose-dependent inhibition of the thrombin enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mechanism of thrombin-induced endothelium-dependent coronary vasodilation in dogs: role of its proteolytic enzymatic activity. 241 89


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