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 previously showed that the alpha-thrombin-antithrombin III complex causes antigenic change in vitronectin as monitored by the monoclonal anti-vitronectin antibody 8E6 (Tomasini & Mosher, 1988). We have extended these studies to other protease-serpin complexes and to gamma-thrombin, a proteolytic derivative of alpha-thrombin. In the presence of heparin, recognition of vitronectin by 8E6 was increased 64- or 52-fold by interaction with the complex of alpha-thrombin and heparin cofactor II or the Pittsburgh mutant (Met358----Arg) of alpha 1-protease inhibitor, respectively. This was comparable to the value obtained with the alpha-thrombin-antithrombin III complex. Factor Xa-serpin complexes were approximately 4-fold less effective than the corresponding thrombin complexes. alpha-Thrombin-serpin complexes but not Xa-serpin complexes formed disulfide-bonded complexes with vitronectin. Antigenic changes and disulfide-bonded complexes were not detected when trypsin- or chymotrypsin-serpin complexes were incubated with vitronectin. gamma-Thrombin caused 7- and 34-fold increases in recognition of vitronectin by MaVN 8E6 in the absence and presence of heparin, respectively. In contrast, alpha-thrombin by itself had no effect. The antigenic change induced by gamma-thrombin was maximal when gamma-thrombin and vitronectin were equimolar, was not dependent on cleavage of vitronectin, and was abolished by inhibition of gamma-thrombin with Phe-Pro-Arg-chloromethyl ketone but not with diisopropyl fluorophosphate. These data indicate that alpha-thrombin is the component in alpha-thrombin-serpin complexes that induces the antigenic change in vitronectin, probably via a region that is preferentially exposed in gamma-thrombin.
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PMID:Conformational lability of vitronectin: induction of an antigenic change by alpha-thrombin-serpin complexes and by proteolytically modified thrombin. 248 65

Employing the known three-dimensional (3D) structure of trypsin, we constructed simple graphics models of human-activated protein C and thrombin catalytic domains. Considering the structural analysis of bovine trypsin and pancreatic trypsin inhibitor complex, the difference of active-site amino acid sequences of human protein C inhibitor and antithrombin III and their inhibitory selectivity toward activated protein C and thrombin, we estimated the enzymatic subsites of activated protein C and thrombin and mapped them on the graphics models. Predicted favorable contacts can explain substrate selectivity of the enzymes. In this study, we used two types of modified ALPHA representations extensively. Since almost no report on the 3D structure of a blood coagulation factor has appeared and even an extensive molecular mechanics or dynamics calculation cannot produce satisfying results, simple graphics representation has several advantages.
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PMID:Mapping active sites of blood coagulation serine proteases--activated protein C and thrombin--on simple graphics models. 248 54

A new water-soluble color reagent, 4-N,N-dimethylaminoazobenzene-4'-isothiocyano-2'-sulfonic acid (S-DABITC), was used to identify lysine residues of antithrombin III which participate in the binding of heparin. Antithrombin, modified with S-DABITC in the presence and absence of low molecular weight heparin (Mr 5000) was reduced, carboxymethylated, and digested with trypsin. The digest was analyzed by high-performance liquid chromatography and monitored at 465 nm. In the absence of heparin, four major colored peptides (T1, T2, T3, and T4) were identified. When antithrombin was preincubated with heparin (2-fold by weight), followed by S-DABITC modification, the recovery of peptide T4 remained unchanged, but the recoveries of T1, T2, and T3 were reduced by 93, 86, and 98%, respectively. In addition, a new colored peptide, TA, appeared. Amino acid sequencing of peptides T1, T2, T3, and TA localized S-DABITC modification sites as Lys-136, Lys-125, Lys-107, and Lys-236, respectively. Thus, binding of heparin to human antithrombin diminished S-DABITC modification at Lys-107, Lys-125, and Lys-136, but at the same time enhanced S-DABITC modification at Lys-236. This phenomenon was further characterized by varying the molar ratio of heparin/antithrombin (from 0.04 to 20). The shielding of Lys-125 and Lys-136 was inversely proportional to the activation of Lys-236. At a heparin/antithrombin molar ratio of 1, the extent of shielding of Lys-125 and Lys-136 and the unmasking of Lys-236 were 25-33%. This shielding-unmasking effect correlated with enhanced antithrombin inhibition of thrombin. We conclude that Lys-107, Lys-125, and Lys-136 are situated within the heparin-binding site of human antithrombin and that binding of heparin to antithrombin causes a conformational change of antithrombin that leads to the exposure of Lys-236 for S-DABITC modification.
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PMID:Binding of heparin to human antithrombin III activates selective chemical modification at lysine 236. Lys-107, Lys-125, and Lys-136 are situated within the heparin-binding site of antithrombin III. 249 30

Pancreatic pseudocyst fluid from eight patients was examined biochemically. The fluid was found to be a mixture of plasma proteins and pancreatic juice, possessing a high proteolytic activity against high- as well as low-molecular-weight proteins. The proteolytic activity was found to be trypsin-, kallikrein- and plasmin-like. Gel filtration studies showed proteolytic activity to be present corresponding to alpha-2-macroglobulin-bound proteases and also to free proteases. Quantitative immunochemical levels were about 30-100% of normal plasma levels for alpha-2-macroglobulin, C1 inhibitor, antithrombin III and alpha-2-antiplasmin. However, there was practically no functional inhibitory capacity left in the pseudocyst fluid, except for alpha-1-protease inhibitor, which retained its inhibitory capacity. Neither native kininogen nor complement factor C3 was found: this was probably a result of the proteolytic activity. It is concluded, that a continuing proteolytic activity within the pseudocyst, although decreasing with aging of the cyst, could explain symptoms and complications caused by the pseudocyst.
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PMID:Pancreatic pseudocyst fluid--a mixture of plasma proteins and pancreatic juice possessing a high proteolytic activity. 253 13

The fibrinolytic enzyme plasmin at 0.25 units/ml produced a contraction of isolated canine basilar arteries that developed slowly and was sustained for at least 2 hours. Plasmin and thrombin (1 unit/ml) acted synergistically to enhance the contractile response. In contrast to plasmin, the marked contraction elicited by thrombin ended within 1 hour, and afterward the artery was completely tachyphylactic to thrombin. Fibrin clot, fibrinopeptides, and fibrin degradation products did not prolong significantly the effect of thrombin or prevent the tachyphylaxis. Plasmin and thrombin may occupy a common membrane receptor because exposing the artery briefly to trypsin (24 micrograms/ml) thereafter abolished the contractile effect of plasmin and thrombin without affecting the action of other agonists. Antithrombin III (1.0 unit/ml) relaxed basilar arteries that were precontracted with plasmin (0.5 unit/ml), thrombin (1.0 unit/ml), serotonin (10(-5) M), uridine triphosphate (10(-4) M), or KCl (8 X 10(-2) M). The results suggest that the vasoconstrictor effect of thrombin might contribute to hemostasis after subarachnoid hemorrhage (SAH) but, because of tachyphylaxis, not to delayed vasospasm. On the other hand, the constrictor action of plasmin might appear late in the course of SAH in association with clot lysis and tissue repair. Last, the level of the vasorelaxant antithrombin III in cerebrospinal fluid could control the appearance and severity of cerebral arterial spasm in SAH.
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PMID:Role of plasmin, thrombin, and antithrombin III as etiological factors in delayed cerebral vasospasm. 257 47

Plasma from insulin-dependent diabetics shows an increased ability to specifically activate the (Na-K)ATPase from different sources. Several protease inhibitors like phenyl methyl sulfonyl fluoride, trypsin inhibitor, antithrombin III and aprotinin, produced a significant dose-dependent inhibition of the stimulatory effect produced by a 1/100 final dilution of plasma on the beef heart (Na-K)ATPase activity. Serine proteases employed at scalar concentrations in the ATPase medium gave a dose-dependent stimulation of the enzyme activity as did diabetic plasma. The maximum percent stimulation of the (Na-K)ATPase activity (about 60%) was reached by 0.56 microgram/ml of thrombin, 0.50 microgram/ml of kallikrein and 0.55 microgram/ml of trypsin. The protease-induced ATPase stimulation was significantly reduced by antithrombin III, trypsin inhibitor and by aprotinin. A partial purification of the activating plasma factor was obtained by eluting plasma on a heparin-Sepharose column. Two (Na-K)ATPase stimulating fractions were found, which eluted with 1.0 and 3.0 mol/l NaCl, respectively. Half-maximal stimulation of the enzyme occurred with 3.4 micrograms/ml proteins of fraction 1.0 mol/l and with 45 ng/ml proteins of fraction 3.0 mol/l, this last representing the most purified plasma fraction (about 8890-fold purification). The proteolytic activity of both plasma and purified plasma fractions was tested on Tos-Arg-OMe substrate which was hydrolyzed to a much higher degree by the most purified plasma fraction. Like the (Na-K)ATPase stimulation, the esterolytic activity was inhibited by protease inhibitors, the most effective to this regard being antithrombin.
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PMID:Identification and partial purification of a (Na-K)ATPase stimulating serine protease from plasma of insulin-dependent diabetics. 283 59

We have isolated three cDNA clones for human alpha 2-plasmin inhibitor (alpha 2-PI). Two clones are from human hepatoma cell line, Hep G2, and cover the entire protein coding region plus the 3'-flanking region up to the poly(A) sequence, and the other clone is from human liver and contains the carboxyl-terminal half. The total length of the cDNAs is 2.29 kb, corresponding to more than 95% of the full-length mRNA. alpha 2-PI seems to consist of 452 amino acid residues plus 39 amino acid residues for the signal peptide. The amino acid sequence shows 23 to 28% homology to those of five other protease inhibitors, plasminogen activator inhibitor (PAI), protein C inhibitor (PCI), alpha 1-antitrypsin (alpha 1-AT), antithrombin III (AT III), and alpha 1-antichymotrypsin (alpha 1-AC). alpha 2-PI seems to be the most distantly related among these inhibitors. Comparison of the phylogenetic trees of proteases and their inhibitors indicates that four proteases, namely elastase (or trypsin), chymotrypsin, plasminogen activator, and thrombin, may have evolved concurrently with the corresponding inhibitors. However, alpha 2-PI and PCI seem to have evolved asynchronously from their substrates. The data suggest that alpha 2-PI may originally have inhibited some protease other than plasmin, and protein C may have had an inhibitor different from the present one early in its evolutionary history.
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PMID:Structure of human alpha 2-plasmin inhibitor deduced from the cDNA sequence. 283 Feb 48

A proteinase inhibitor has been isolated from human colorectal adenocarcinomas by extraction with a low-ionic-strength buffer and a combination of Con A-Sepharose, Sephadex G-200, DEAE-cellulose and chromatofocusing steps. The preparation appeared to be homogeneous upon gel exclusion chromatography and SDS-polyacrylamide gel electrophoresis and had an estimated molecular weight of 66,000. The inhibitor was able to bind and inhibit urokinase, plasmin, trypsin, tissue plasminogen activator and thrombin. The binding appeared to be stoichiometric and relatively fast. The isoelectric point of the protein was 4.6-4.7. The inhibitor did not crossreact with antisera elicited against alpha 2-macroglobulin, alpha 2-antiplasmin, antithrombin III or C1-inhibitor, but it did crossreact with an antiserum against alpha 1-antitrypsin in double immunodiffusion. The antiserum only partially attenuated the activity of the inhibitor. Whereas alpha 1-antitrypsin completely inhibited the amidolytic activity of elastase, the tumor inhibitor had no effect on elastase under the same conditions.
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PMID:Isolation and partial characterization of a proteinase inhibitor from human colorectal adenocarcinoma. 293 82

The purpose of this study was to investigate the relation between serum lipase (LP), serum immunoreactive trypsin (IRT), and its inhibitors in patients with adult respiratory distress syndrome (ARDS) of diverse origin and to compare their time course with other acute conditions. The IRT and LP levels were determined at regular intervals in 41 patients hospitalized in the intensive respiratory unit with ARDS (n = 9), acute pancreatitis (n = 5), shock (n = 9), bronchopneumonia (n = 10), or acute cardiogenic pulmonary edema (n = 8). Several trypsin inhibitors were measured simultaneously: serum trypsin inhibitory capacity (TIC), alpha 1-antitrypsin, alpha 2-macroglobulin, and antithrombin III. Concomitantly, angiotensin-converting enzyme (ACE) activity was determined as a potential marker of the endothelial injury. A respective 19- and 13-fold increase in IRT and LP values were observed in patients with ARDS after a mean evolution of 6 days; similar increases were seen in patients with pancreatitis. These values were significantly higher than those observed in the other conditions studied. In patients with ARDS and acute pancreatitis, the evolution of IRT and LP values were associated with a sixfold rise in TIC. A low TIC/IRT ratio in patients with ARDS appeared to be an index of poor prognosis. Conversely, ACE activity evolution was characterized by an early decrease in all the conditions studied. These observations indicate that there is an acute delayed pancreas injury in ARDS. Thus, the release of pancreatic enzymes are not reliable markers of the early evolution of the disease but they may represent secondary mediators for enhancement of the increased endothelial permeability.
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PMID:Evidence for pancreas injury in adult respiratory distress syndrome. 298 86

A trypsin-like enzyme (tryptase) has been purified to homogeneity from the granules of a human cytolytic lymphocyte (CTL) line, Q31, by a three-step procedure. By including 0.3% (v/v) Triton X-100 and 1 mg/ml heparin in purification buffers, near total yields of tryptase activity were obtained during the purification. The enzyme, referred to as Q31 tryptase, migrated in polyacrylamide gels with sodium dodecyl sulfate at a position corresponding to 28 kDa with and to 45 kDa without 2-mercaptoethanol. It had an amino-terminal sequence identical to a previously reported human CTL tryptase at 20 of 22 positions identified. It hydrolyzed N alpha-carbobenzyloxy-L-lysyl-thiobenzyl ester (BLT), and this BLT esterase activity was most efficient at slightly alkaline pH and was relatively more active near neutral pH than mouse CTL tryptase. Human alpha 1-protease inhibitor, human antithrombin III, phenylmethanesulfonyl fluoride, and p-aminobenzamidine inhibited the Q31 tryptase. The inhibition by human antithrombin III was rapid enough to be of physiological significance. A survey of oligopeptide p-nitroanilides found that the best substrate for human Q31 tryptase is H-D-(epsilon-carbobenzyloxy)Lys-L-Pro-L-Arg-p-nitroanilide. The Q31 tryptase appears to have broad specificity for amino acid residues at P2 and P3, i.e. at 2 and 3 residues amino-terminal to the scissile bond.
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PMID:Human cytotoxic lymphocyte tryptase. Its purification from granules and the characterization of inhibitor and substrate specificity. 304 19


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