Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT02079 (lysine)
 58,762 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

alpha- and beta-Fibrinogenases (EC 3.4.21.5) were purified from Trimeresurus mucrosquamatus venom by the technique of recycling chromatography. Both enzymes were single polypeptide chains and homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and ultracentrifugation. The sedimentation constants of alpha- and beta-fibrinogenases were 2.52 and 3.04 respectively. The molecular weight of alpha-fibrinogenase was 21 500--23 400, and that of beta-fibrinogenase was 25 000--26 000. The contents of proline, glycine and tryptophan were higher in beta-fibrinogenase than in alpha-fibrinogenase. The isoelectric points of alpha- and beta-fibrinogenases were pH 8.1 and 5.7 respectively. The optimal pH of alpha-fibrinogenase was about 7.4 and that of beta-fibrinogenase was around 8.5. The activity of alpha-fibrinogenase was completely destroyed after 30 min at 60 degrees C, pH 5.6, 7.4 and 9.0, while that of beta-fibrinogenase was not significantly affected by the same treatment. Both enzymes showed proteolytic activities toward fibrinogen and casein, but were devoid of phospholipase A, alkaline phosphomonoesterase and phosphodiesterase activities of the crude venom. The tosyl-L-arginine methylester esterase activity of beta-fibrinogenase was about 17 times that of the crude venom, while alpha-fibrinogenase was completely devoid of this activity. The fibrinogenolytic activity of alpha-fibrinogenase was markedly inhibited by EDTA and cysteine, while that of beta-fibrinogenase was inhibited markedly by phenylmethane sulfonylfluoride and slightly by tosyl-L-lysine chloromethylketone and cysteine.
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PMID:Physicochemical properties of alpha- and beta-fibrinogenases of Trimeresurus mucrosquamatus venom. 1 16

Staphylokinase (SAK)-activated plasminogen reacted specifically with nitroanilide (N-benzoyl-DL-lysine-4-nitroanilide) to form intensively yellow 4-nitroaniline. This reaction did not occur with staphylococcal proteases. For qualitative SAK-determinations nitroanilide was incorporated in an agar medium. SAK diffused into the medium and caused a distinct change in color from white to yellow. For quantitative SAK-determination the conversion of colorless nitroanilide to yellow 4-nitroaniline was recorded photometrically at 405 nm. The optical density correlated well with SAK-activity of preparations with different degrees of purity (fig. 1, 2). Another quantitative procedure for SAK-activity could be conducted with fibrin in microtiter-plates (Fib-MTT). In this method, after 2-fold dilutions of SAK-preparations fibrinogen (stained blue with astrazonblue) and subsequently thrombin were added. SAK-activity was indicated by lysis of the blue-colored fibrin clots in the microtiter-plates (fig. 3). The Fib-MTT was particularly suitable for measuring wide ranges of SAK-activities.
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PMID:[Qualitative and quantitative determinations of staphylokinase-activity (author's transl)]. 21 37

Present methods for assay of platelet aggregating agents use freshly prepared platelets. Much time is spent in daily preparation of platelets and standardization presents problems. The preparation of fixed washed platelets (FWP) and their use in two bioassays are described in this report. Washed human platelets were fixed for 48 hours with 4 per cent paraformaldehyde, washed twice in phosphate buffer, pH 6.4, and stored at 4 degrees C. Aggregation of FWP was studied with a macroscopic test and a light absorbance measurement. FWP did not aggregate with adenosine diphosphate, collagen, adrenalin, and thrombin. FWP aggregated with bovine or porcine plasma, poly-L-lysine, and ristocetin with normal human plasma but not with von Willebrand's disease plasma. These observations confirm the direct aggregating effect of these agents. Macroscopic aggregation times were dependent on the amount of aggregating agent (bovine plasma, normal human plasma). A quantitative assay for bovine platelet aggregating factor (PAF) and von Willebrand factor (vWF) with FWP was developed. The ability of FWP to aggregate remained unchanged after 1 month of storage at 4 degrees C. Ristocetin alone caused a decrease in light transmission of FWP suspensions, depending upon the concentration of ristocetin, but did not cause aggregation. FWP constitute a stable reagent suitable for quantitative measurement of PAF and vWF.
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PMID:Platelets fixed with paraformaldehyde: a new reagent for assay of von Willebrand factor and platelet aggregating factor. 23 99

1. Beta-Phenylpropionylthiocholine and N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide (dansylcadaverine) serve as a pair of water-soluble (pH7.5) model substrates for transamidating enzymes. Amide formation could be followed directly through fluorescence measurements by monitoring the continuous extraction of the water-soluble coupling product, N-(beta-phenylpropionyl)dansylcadaverine, into n-heptane. By this procedure, the steady-state kinetics of glutamine-lysine endo-gamma-glutamyltransferase from human plasma (fibrinoligase, thrombin- and Ca2+-activated blood coagulation Factor XII) and from guinea-pig liver (liver transglutaminase) were investigated at 25 degrees C. 2. With beta-phenylpropionylthiocholine as the varied substrate, Lineweaver-Burk plots with various concentrations of dansylcadaverine intercept on the horizontal axis, suggesting that formation of the acyl-enzyme is rate limiting. 3. On the basis of functional normality of active sites, kcat. values of 1.8 s(-1) and 0.9 s(-1) were obtained for the plasma and liver gamma-glutamyltransferase respectively. The two enzymes show identical affinities for the first substrate, beta-phenylpropionylthiocholine, with Ka 4 times 10(-4) M. 4. Utilization of the second substrate, dansylcadaverine, appears to be an order of magnitude more efficient with the liver enzyme. 5. N-(5-Amino-3-thiapentyl)-5-dimethylaminonaphthalene-1-sulphonamide (dansylthiacadaverine) could be used instead of dansylcadaverine in the fluorescent kinetic system. 6. Competitive inhibition by a non-fluorescent amine substrate histamine was also evaluated.
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PMID:Transamidase kinetics. Amide formation in the enzymic reactions of thiol esters with amines. 23 98

The partial covalent structure of bovine beta-thrombin has been determined by the use of automated Edman degradation and carboxypeptidase digestion of the component polypeptide chains separated by gel filtration following either reduction and carboxymethylation or performic acid oxidation. beta-Thrombin has been found to contain three peptide chains derived by proteolysis of the parent alpha-thrombin molecule. The A chain of alpha-thrombin has been cleaved at two points yielding a peptide (A1 chain) which contains 17 amino acids, beginning with threonine 14 and ending with lysine 30. The B chain of alpha-thrombin has been cleaved at two positions to yield a B1 chain which begins with the NH2-terminal isoleucine and terminates with lysine 65 and a B2 chain which begins with lysine 74 and continues through COOH-terminal serine 259. The A1 chain and B2 chain are linked by a disulfide bridge. Although there is no evidence for a covalent bond between the B1 chain and the B2-A1 chains, the B1 chain is tightly bound to the remainder of the molecule, for separation is achieved only under denaturing conditions.
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PMID:The covalent differences between bovine alpha- and beta-thrombin. A structural explanation for the changes in catalytic activity. 46 39

The effect of intravenous administration of homologous fibrin degradation products and thrombin on fibrinogen synthesis was assessed in rabbits. The relative fibrinogen synthesis rate was calculated as a ratio of the amount of radiolabelled lysine incorporated into fibrinogen to the amount incorporated into albumin during the same measurement period. An increase in this ratio above control would indicate a relatively specific stimulation of fibrinogen synthesis as compared with albumin, which is not an acute-phase reactant. Injection of 45 mg of 'early' or 'late' fibrin degradation products failed to produce a significant increase in the relative fibrinogen synthesis rate, suggesting that fibrin degradation products play no feedback role in controlling fibrinogen synthesis. Infusion of small amounts of homologous thrombin (15--25 NIH u) was followed by a small but statistically significant elevation of the relative fibrinogen synthesis rate. This was not accompanied by any increase in the levels of fibrinogen degradation products in plasma, or by any decrease in plasma fibrinogen concentration, possibly suggesting that thrombin can stimulate fibrinogen synthesis by a mechanism independent of significant fibrinogenolysis or intravascular coagulation.
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PMID:Effect of fibrin degradation products and thrombin on fibrinogen synthesis. 52 46

Throbin-activated human platelets cause agglutination of trypsinized, formalinized bovine erythrocytes. This lectin activity of stimulated platelets was blocked by galactosamine, glucosamine, mannosamine, lysine, and arginine, but not by N-acetylated sugars, other neutral sugars, or other amino acids. Inhibitors of the thrombin-induced lectin activity also blocked thrombin-induced platelet aggregation. It appears that a membrane surface component that has lectin activity mediates platelet aggregation.
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PMID:Thrombin-induced platelet aggregation is mediated by a platelet plasma membrane-bound lectin. 66 8

It was recently demonstrated that C-reactive protein (CRP)4 inhibits the response of human platelets to heataggregated human gamma-globulin and thrombin and that this inhibition is characterized by a dose-dependent reduction in aggregation, activation of platelet factor 3 (PF3), and release of beta-glucuronidase. In the present experiments, CRP was found also to inhibit the ability of washed human platelets to aggregate in response to poly-L-lysine (PLL); in these experiments, the magnitude of the inhibitory effect was dependent upon the m.w. of PLL used as the stimulating agent, and was more effective with low (15,000 daltons) than with high (400,000 daltons) m.w. polymers. CRP similarly inhibited ADP- and epinephrine-stimulated platelet aggregation in platelet-rich plasma (PRP), and this was characterized by relatively minimal suppression of the primary wave of aggregation. CRP also inhibited the platelet aggregation induced by collagen in PRP, although it had no effect upon the adherence of platelets to collagen. Finally, CRP inhibited the activation of PF3 and the release of serotonin during stimulation of platelets with ADP, and this inhibition was temporally related to the onset of the secondary wave of aggregation. These experiments extend the platelet reactivities inhibited by CRP, show that CRP expresses its inhibitory capacity in platelet-rich plasma as well as upon isolated platelets, raise the possibility that CRP exercises its effects by inhibiting or interfering with the release and/or utilization of endogenous platelet ADP, and support the concept that CRP plays an important role in the control of platelet responsiveness to a variety of stimuli during acute inflammatory reactions.
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PMID:Effects of C-reactive protein on platelet function. II. Inhibition by CRP of platelet reactivities stimulated by poly-L-lysine, ADP, epinephrine, and collagen. 97 42

Partially purified bovine prothrombin was activated in half-saturated trisodium citrate seeded with thrombin, and the resulting thrombin was chromatographed on Amerblite IRC-50, followed by rechromatography on DEAE-Sephadex A-50. Five fractions, possessing both esterase and clotting activities, were partially isolated, but fraction VI was shown to be a pure three-chain active species with threonine, isoleucine and lysine, in 1:1:1 molar proportions as N-termini. The amino acid composition and C-termini of fraction VI were determinied. The molecular weights of the isolated chains, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, were 7300, 12000 and 19500 respectively. These data, when taken together with the amino acid sequence of the two-chain thrombin reported by Magnusson et al. (1975) [in Prothrombin and related Coagulation Factors, (Hember, H. C. & Veltkamp, J. J., eds.), pp. 25-46, Leiden University Press, Leiden], indicated that proteolysis occurred at the Arg(78)-Lys(79) peptide bond of the B chain of a precursor molecular species, thus converting this two-chain species into the three-chain active form described here.
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PMID:Isolation and characterization of an active three-chain molecular species of bovine thrombin. 99 41

Both the clotting and esterase activities of thrombin are inhibited by alpha1-proteinase inhibitor (alpha1-antitrypsin). The inhibition is a time-and temperature-dependent reaction which is proportional to the molar ratio of thrombin to inhibitor. Both the active-site serine residue of thrombin and the reactive-site lysine residue of alpha1-proteinase inhibitor are involved. alpha1-Proteinase inhibitor forms a 1:1 complex with thrombin that is comparable with the complex formed with trypsin and other proteinases. Incubation of the inhibitor with excess of thrombin, however, results in inactivation of nearly all the enzyme, even though only as much complex is formed as alpha1-proteinase inhibitor present. A portion of the remaining thrombin apparently aggregates. These results suggest that the mechanism for inhibition of thrombin may not be exactly the same as for trypsin, which is inhibited only to the extent to which complex is formed.
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PMID:Inactivation of human thrombin in the presence of human alpha1-proteinase inhibitor. 108 57


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