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:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two arginine ester hydrolases, designated EI and EII, consist of multiple molecular forms with pI values in the range 4.0-4.6 for EI and 3.3-3.9 for EII. Isoforms had identical molecular weights: 38,500 for EI and 41,000 for EII (SDS electrophoresis). The N-terminal amino acid for both enzymes was valine and their amino acid contents were very similar, with both containing carbohydrate. After treatment of EI and EII with neuraminidase both enzymes migrated identically in the electrofocusing system. Neither esterase hydrolyzed casein, alpha-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA), yet both hydrolyzed alpha-N-benzoyl-L-arginine methylester (BAEE), p-tosyl-L-arginine methylester (TAME) and Pro-Phe-Arg-MCA. The esterase activities of the two enzymes were inhibited by organophosphorus inhibitors and benzamidine. The Km value for EI with BAEE was 3.3 X 10(-5) M, with TAME 3.0 X 10(-5) M, and for EII 2.7 X 10(-5) M (BAEE) and 5.9 X 10(-5) M (TAME). EII possessed kinin-releasing activity, as shown by the twitch response of an isolated rat uterus. The physiological role of EI is unknown. Neither esterase has thrombin-like or fibrionlytic activities.
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PMID:Purification and characterization of two arginine ester hydrolases from Vipera berus berus (common viper) venom. 361 75

By means of CM-Sephadex C-50 column chromatography and gel filtration on Sephadex G-75 and G-50 columns, a potent platelet aggregation inhibitor was purified and characterized. It was a glycoprotein with a molecular weight of 31,000. It was devoid of phospholipase A, ADPase, esterase and fibrino(geno)lytic activities. It inhibited dose-dependently the aggregation of washed platelets induced by collagen, thrombin, sodium arachidonate, platelet activating factor and ionophore A23187 with a similar IC50 (5-10 micrograms/ml). It was also active in platelet-rich plasma, with an IC50 of 10-15 micrograms/ml. The venom inhibitor reduced the elasticity of whole blood clot and inhibited the thrombin-induced clot retraction of platelet-rich plasma. These activities were related to its inhibitory activity on platelet aggregation rather than blood coagulation. The venom inhibitor had various effects on [14C]serotonin release stimulated by aggregation agonists. It had no effect on thromboxane B2 formation of platelets stimulated by sodium arachidonate, collagen and ionophore A23187. The presence of this venom inhibitor prior to the initiation of aggregation was a prerequisite for the maintenance of its maximal activity. It showed a similar inhibitory effect on collagen or thrombin-induced aggregation even when it was added after the platelets had undergone the shape change. High fibrinogen levels partially antagonized its activity. The venom inhibitor completely inhibited the fibrinogen-induced aggregation of alpha-chymotrypsin-treated platelets. It is concluded that this venom inhibitor interferes with the interaction of fibrinogen with fibrinogen receptors, leading to inhibition of aggregation.
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PMID:Characterization of a potent platelet aggregation inhibitor from Agkistrodon rhodostoma snake venom. 362 Apr 99

This report documents attempts to mimic the rate enhancement effect of thrombomodulin on human alpha-thrombin-catalyzed activation of human protein C in the absence of exogenous calcium. Specifically the following tryptamine analogs at 1 mM concentration were shown to enhance the protein C activation rate relative to a control with no added effector at pH 8.3 (50 mM Tris-HCl, 0.1 M NaCl, 37 degrees C): serotonin, 1.2; tryptamine, 2.9; 5-fluorotryptamine, 4.4; 6-fluorotryptamine, 7.2. At much higher levels, e.g. 10 mM, all of the above effectors, as well as indole, showed a moderate inhibition of human protein C activation. ATP, a platelet release product, showed a sigmoidal inhibition pattern similar to that found previously for thrombin amidase, clotting, and esterase activity (Conery, B.G., and Berliner, L.J. (1983) Biochemistry 22, 369-375). Overall, the enhancement factors for human alpha-thrombin activation of protein C with the tryptamine analogs described above were remarkable when considering the effect of a simple ligand versus the natural activator, thrombomodulin.
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PMID:Ligands which effect human protein C activation by thrombin. 365 43

The activation of prothrombin has been studied by using highly purified preparations of activated factor X1 and activated factor X2, factor V and prothrombin. The rate of prothrombin activation was followed using an esterase assay involving the conversion of N alpha-benzoylarginine ethyl ester (BAEE) by thrombin generated in the course of prothrombin activation. The rate of thrombin generation increased by about 26000-fold when factor V and phospholipid were added to prothrombin, factor Xa and calcium. A comparison of the rates of thrombin formation obtained with activated factor X1 and activated factor X2 showed that activated factor X1 had only 70% of the biological activity of activated factor X2. Attempts to explain the rate of prothrombin activation and the difference between the activity of activated factor X1 and activated factor X2 are discussed.
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PMID:Activation of prothrombin as measured by the conversion of Nalpha-benzoylarginine ethyl ester. 374 19

It was shown that selective hydrolysis of the disulfide bridge between the A- and B-chains of human thrombin in the absence of denaturating agents decrease its proteolytic (e.g., fibrinogen-binding), esterase and amidase activities. Both chains remain bound by non-covalent interactions. A preparation of partially reduced thrombin was obtained and its kinetic parameters were determined. The experimental results suggest that the S-S bond connecting the A- and B-chains of thrombin is involved in the stabilization of the enzyme active center.
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PMID:[Effect of reduction of the interchain disulfide bridge of human thrombin on its enzymatic activity and structure]. 377 71

Successive thrombin modification by carbodiimide and aliphatic diamines decreases esterase and fibrin-coagulating activity of the enzyme. Modified thrombin causes no platelet aggregation. Water-soluble enzyme conjugates devoid of fibrinogen-coagulating action and possessing increased fibrinolytic affinity to the site of fibrin clot location have been obtained by covalent binding of chymotrypsin to modified thrombin.
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PMID:[Fibrinolytic action of an enzyme preparation covalently bound to modified thrombin]. 380 48

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

An enzyme bearing thrombin-like specificity has been purified to homogeneity from the venom of Trimeresurus flavoviridis (the Habu snake). The enzyme is a monomer with a molecular weight of 23,500 as determined by analytical gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein contains approximately 210 amino acid residues and has a relatively high content of aspartic acid and glutamic acid. The isoelectric point was 4.8 and the extinction coefficient at 280 nm for a 1% solution was 11.5. The enzyme acted directly on fibrinogen to form a fibrin clot with 2.0 NIH units. Analysis by high performance liquid chromatography of enzyme-treated fibrinogen revealed the release of a peptide identical in composition to thrombin-induced fibrinopeptide A, but no peptide corresponding to fibrinopeptide B was detected. The enzyme showed esterase and amidase activities on synthetic substrates containing arginine. The enzyme exhibited higher activity toward tosyl-L-arginine methyl ester (TAME) but 6-times lower activity toward benzoyl-L-arginine p-nitroanilide when compared with bovin thrombin. The esterase activity was inhibited by diisopropylfluorophosphate and at a slower rate by phenylmethanesulfonyl fluoride, but was least affected by tosyl-L-lysine chloromethyl ketone, showing that the enzyme is a serine protease like thrombin. The enzyme showed a bell-shaped pH dependence of kcat/Km for hydrolysis of TAME, with a maximum around pH 8.5.
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PMID:Purification and characterization of a coagulant enzyme from Trimeresurus flavoviridis venom. 391 Jun 43

Echis carinatus venom was separated into twenty fractions by means of ultrafiltration and CM-Sephadex C-50 column chromatography. Fraction II possessed inhibitory activity on the aggregation of washed rabbit platelets and fraction XII possessed the procoagulant and platelet aggregation-inducing activity. Both were further purified by gel filtration on a Sephacryl S-200 column. The purified aggregation inducer was a glycoprotein with procoagulant activity 10-12-times that of the crude venom. It possessed proteinase and amidase but was devoid of esterase activity. The molecular weight was 16 000, and it contained 8.7% of neutral sugar. The isoelectric point was pH 7.6. The purified aggregation inhibitor was a single peptide chain with a molecular weight of 6800 and contained 22.1% of neutral sugar. The isoelectric point was pH 4.8. It was devoid of any enzymatic activity of the crude venom. The IC50 was about 10 micrograms/ml on the thrombin-induced platelet aggregation. The inhibitory activity was fully retained after the treatment of the venom aggregation inhibitor with neuraminidase, but was completely destroyed by sodium metaperiodate. Upon heat treatment at 90 degrees C, the venom aggregation inhibitor was heat stable at pH 5.5 for 4 h, but was completely destroyed after 2 h at pH 8.9 and retained about 50% of its inhibitory activity of the control at pH 7.2 for 4 h. The venom aggregation inhibitor decreased the elasticity of the whole blood clot, and this effect was related to its inhibitory action on platelet aggregation instead of blood coagulation.
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PMID:Characterization of the platelet aggregation inducer and inhibitor from Echis carinatus snake venom. 401 43

Two arginine ester hydrolases, designated AAEI and AAEII, from the venom of Crotalus scutulatus scutulatus have been investigated. The amino acid content of both enzymes were very similar and both esterases contained carbohydrate. Following treatment of AAEI and AAEII with neuraminidase, both enzymes migrated identically in two electrophoresis systems and one electrofocusing system. The esterase activities of both enzymes were optimally active in the range pH 8.0-8.5. Neither esterase hydrolyzed casein, hemoglobin (Hb) or alpha-N-benzoyl-DL-arginine-p-nitroaniline (BAPNA), yet both AAEI and AAEII hydrolyzed alpha-N-benzoyl-L-arginine ethyl ester (BAEE), alpha-N-benzoyl-L-arginine methyl ester (BAME), p-tosyl-L-arginine methyl ester (TAME) and acetylphenylalanylarginine methyl ester (Ac-Phe-Arg-OMe). The esterase activities of the two enzymes were inhibited by serine specific reagents and benzamide, but not by EDTA or soybean trypsin inhibitor. The Km values for each enzyme with alpha-N-benzoyl-L-arginine ethyl ester and acetylphenylalanylarginine methyl ester were determined. Neither esterase displayed thrombin-like or fibrinolytic activities. Both AAEI and AEII possessed kinin releasing activity as shown by the twitch response of an isolated rat uterus. The N-terminal sequences of AAEI and AAEII were identical and both enzymes sequences were similar to other arginine esterases from crotalid venoms. The properties of AAEI and AAEII are compared to several other arginine esterases possessing kallikrein-like activities which have been isolated from snake venoms.
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PMID:Characterization of two arginine ester hydrolases from Mojave rattlesnake (Crotalus scutulatus scutulatus) venom. 402 35


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