Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.4 (deaminase)
 5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An enzyme displaying proteolytic activity toward the natural substrate casein as well as clotting activity on fibrinogen was purified to homogeneity from Cerastes cerastes (horned viper) venom and characterized. The enzyme is constituted of two identical subunits of mol. wt 48,500 as determined by SDS-polyacrylamide gel electrophoresis, and has an isoelectric point of 3.75. N-terminal sequencing up to the 33rd residue evidenced a high homology with other snake venom proteinases. The proteinase is of serine-type as indicated by high sensitivity to DFP and shows both arginine-ester hydrolase and amidase activities on synthetic substrates. Both specific activities were 30-fold higher than the respective activities found in the crude venom. The Km value determined for arginine-containing substrate BAEE was 3.0 x 10(-4) M and the Km for chromogenic substrate CBS 34-47 0.65 x 10(-4) M. The Vm/Km ratio, however, was two-fold higher for BAEE than for CBS 34-47; the arginine-esterase activity of this enzyme is thus slightly higher than its amidase activity.
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PMID:A fibrinogen-clotting serine proteinase from Cerastes cerastes (horned viper) venom with arginine-esterase and amidase activities. Purification, characterization and kinetic parameter determination. 148 36

Kinetic studies of the inhibition of thrombin amidase activity by recombinant hirudin have been conducted as a function of salt concentration in the range 0.05 to 1 M, using NaCl, KCl, NaBr and KBr. At the same ionic strength, the value of KI for thrombin-hirudin interaction is found to be different with different salts. The slope d ln KI/d ln a+/-, where a+/- is the mean ion activity, is constant in the range 0.05 to 0.5 M, is sensitive to the particular salt present in solution and is equal to 1.07 +/- 0.09 (NaCl), 0.92 +/- 0.10 (KCl), 1.37 +/- 0.10 (NaBr) and 0.56 +/- 0.10 (KBr). These results indicate that specific ion effects are involved in the modulation of thrombin-hirudin interaction in the form of ion release, as recently found in the case of thrombin interaction with its natural substrate fibrinogen. The linkage hierarchy for ion release found in the case of thrombin-fibrinogen interaction also applies in the case of thrombin-hirudin interaction, with the number of released ions decreasing in the order NaBr greater than NaCl greater than KCl greater than KBr. It is proposed that the process of bridge-binding to the fibrinogen recognition site and the catalytic pocket of the enzyme, as seen in the case of fibrinogen and hirudin, is linked to ion release and controlled by modulation of the association rate constant.
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PMID:Modulation of thrombin-hirudin interaction by specific ion effects. 161 55

A model-independent (phenomenological) characterization of the clotting curve is proposed. Three parameters are used to encapsulate the main features of the increase in absorbance observed at 350 nm due to the reaction of thrombin with fibrinogen that leads to clot formation: (1) the maximum increase in absorbance per unit time, delta Am, at the inflection point of the clotting curve; (2) the time needed to reach the maximum increase in absorbance, tm; and (3) the clotting time, tc, obtained from extrapolation of the slope at tm to the zero absorbance baseline. Clotting curves at low fibrinogen concentrations (0.125 divided by 0.250 microM), well below the Km, where thrombin amidase activity is rate-limiting with respect to the subsequent aggregation process, have been measured under a wide variety of experimental conditions, (i.e., as a function of thrombin concentration, pH and temperature) in order to explore the basic response of each parameter to changes in solution conditions. Under all conditions examined in this study we have observed that tm and tc are linked through a linear relationship that appears to be an important invariant property of the clotting curve, regardless of experimental conditions. No such clear relationship exists between delta Am and tc, with tc being associated with several possible values of delta Am and vice versa, depending upon solution conditions. It is proposed that tc is strictly dependent on thrombin amidase activity, while delta Am reflects properties of the aggregation process leading to clot formation. The clotting time shows a pH and temperature dependence that closely resembles that of Km/Vm for synthetic amide substrates. Furthermore, tc changes linearly with either the inverse thrombin concentration and the concentration of competitive inhibitors of fibrinogen binding to thrombin, as expected for the ratio Km/Vm. We show how the analysis of clotting curves obtained at different thrombin and inhibitor concentrations yields a quantitative measure of KI that is in excellent agreement with the value determined independently from steady-state measurements of thrombin amidase activity.
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PMID:Phenomenological analysis of the clotting curve. 179 5

The A- and B-chains have been isolated from the non-covalent complex of human thrombin A- and B-chains, using selective reduction of the interchain disulfide bridge. The B-chain thus isolated (de-A-thrombin) retains its conformation, which is close to the native one and thus differs considerably from the B-chain isolated from the fully reduced enzyme. Nevertheless, the proteolytic (in terms of fibrinogen clotting) and amidase activities of de-A-thrombin are markedly reduced as compared to the native enzyme and the non-covalent complex of A- and B-chains. It is assumed that the A-chain of thrombin is necessary for normal functioning of the active site of thrombin localized in the B-chain.
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PMID:[Enzymatic properties of the isolated B-chain of human thrombin]. 275 67

A covalent conjugate between the plasminogen activator urokinase and polyclonal rabbit anti-human fibrinogen has been formed using the heterobifunctional coupling reagent N-succinimidyl 3-(2-pyridyldithio) propionate. The resultant urokinase-anti-human fibrinogen conjugate was separated from unreacted material by gel filtration. The conjugate exhibited amidase activity against the small chromogenic substrate pyroglutamyl-glycyl-arginine-p-nitroanilide as well as plasminogen activator activity in an assay employing plasminogen and the plasmin substrate D-valyl-leucyl-lysine-p-nitroanilide. Retention of antibody specificity for fibrinogen was demonstrated using an enzyme linked immunoassay procedure. The conjugate was found to have greater stability in human plasma than unconjugated urokinase.
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PMID:Plasminogen activator-anti-human fibrinogen conjugate. 316 1

Fibrinogen, the major structural precursor of blood clots, was deglycosylated by peptide-N-(N-acetyl-beta-glucosaminyl)asparagine amidase without denaturation of the polypeptide chains. Deglycosylated fibrinogen behaved normally in clinical coagulation assays, although it is less soluble than normal fibrinogen. However, the turbidity of clots formed from deglycosylated fibrinogen always rose faster and higher than that of clots from normal fibrinogen. Scanning and transmission electron microscopy demonstrated that fibrin made from clots of deglycosylated fibrinogen consisted of thicker, less-branched fiber bundles in a more porous network. Moreover, the degree of lateral aggregation was directly related to clot turbidity and inversely related to branching. Deglycosylation promoted turbidity development, lateral aggregation, and porosity of clots under all conditions tested. All other steps in the coagulation pathways appeared to be unaffected by the absence of carbohydrate. These results suggest that carbohydrate constitutively affects the behavior of deglycosylated fibrinogens by 1) contributing a repulsive force that promotes fibrinogen solubility and limits fibrin assembly and 2) sensitizing fibrin to conditions that influence assembly and clot structure.
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PMID:Deglycosylation of fibrinogen accelerates polymerization and increases lateral aggregation of fibrin fibers. 317 May 75

gamma-Thrombin was produced during autolysis or limited proteolysis of coagulant gamma-thrombin. This thrombin form loses its ability to coagulate fibrinogen but preserves the esterase and amidase activity on the low-molecular-weight synthetic substrates. This evidences for the integrity of the active site of gamma-thrombin and for the integrity break of the enzyme molecule region responsible for the binding with fibrinogen. gamma-Thrombin with the minimal coagulating activity, possessing high esterase and amidase activity is obtained. Fibrin-agarose possessing affinity to gamma-thrombin and specifically not binding gamma-thrombin was used to remove admixtures of the coagulant gamma-thrombin from the preparations of gamma-thrombin obtained during the enzyme autolysis.
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PMID:[Affinity chromatography of human gamma-thrombin on fibrin-agarose]. 318 51

A thrombin-like enzyme was isolated in 6% yield from the venom of Crotalus durissus terrificus by ammonium sulfate precipitation followed by gel filtration on Sephadex G-75 and finally affinity chromatography on Sepharose-1,4-butanediol-diglycyl-p-aminobenzamide eluted with 0.15 M benzamidine. The enzyme behaved like a single component on SDS-PAGE corresponding to a molecular weight of 34 kDa. The specific activity of the enzyme toward bovine fibrinogen was 71 NIH U/mg protein. The pH optimum for the coagulation of human fibrinogen was 8.0. The enzyme hydrolyzes the alpha-chain of fibrinogen, has amidase activity on L-arginine-p-nitroanilide and L-arginine-7-amido-4-methyl-coumarin amino terminal blocked peptides and presents esterolytic activity on N-alpha-tosyl-L-arginine-methylester.
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PMID:Isolation and characterization of a thrombin-like enzyme from the venom of Crotalus durissus terrificus. 359

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

Unsulfated N alpha-acetyl-hirudin45-65 (MDL 27 589), which corresponds to the C-terminus of hirudin1-65, was synthesized by solid-phase methods. The synthetic peptide was able to inhibit fibrin formation and the release of fibrinopeptide A from fibrinogen by thrombin. The catalytic site of thrombin was not perturbed by the synthetic peptide as H-D-Phe-Pip-Arg-pNA hydrolysis (amidase activity) was not affected. The binding of synthetic peptide and thrombin was assessed by isolation of the complex on gel-filtration chromatography. A single binding site with a binding affinity (Ka) of approx. 1.0 X 10(5) M-1 was observed for thrombin-hirudin45-65 interaction. The data suggest that the C-terminal residues 45-65 of hirudin contain a binding domain which recognizes thrombin and yet does not bind to the catalytic site of the enzyme.
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PMID:Antithrombin properties of C-terminus of hirudin using synthetic unsulfated N alpha-acetyl-hirudin45-65. 380 84


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