David P. Kosow

Evidence that human α-thrombin is a monovalent cation-activated enzyme

Abstract The activity of human α-thrombin (EC 3.4.21.5) on small peptide substrates was enhanced by NaCl or KCl while tetramethylammonium chloride ((CH 3 ) 4 NCl) or choline chloride (HO(CH 2 ) 2 N(CH 3 ) 3 Cl) which were used as ionic strength controls were without effect. The steady-state kinetic parameters of thrombin amidolysis of several peptidyl p -nitroanilide substrates were measured. Na + enhanced thrombin activity by decreasing the K m,app (0.2 to 0.7-fold) of all substrates, as well as increasing thombin turnover (3.4 to 4.5-fold) of some substrates. The average K A for Na + for the four substrates examined was 3.5 × 10 −2 m . A comparison of the effects of Na + vs K + on thrombin hydrolysis of a single substrate indicated that both cations similarly decreased the K m,app (0.2 to 04.-fold) and increased the k cat,app (3.1 to 3.4-fold) except that higher K + concentrations ( K A = 2.8 × 10 −1 M) were required. The rate of inactivation of thrombin by the active site-directed inhibitor N-p -tosyl-lysine chloromethyl ketone under pseudo-first-order conditions was enhanced 3-fold by saturating NaCl. Also, the fibrinogen clotting activity of thrombin was enhanced by NaCl compared to the choline chloride control. Spectral studies demonstrated that thrombin titration by Na + caused a positive ultraviolet difference spectrum with maxima at 281.5 and 288.5 nm (Δϵ 288.5 = +1067). The K m for Na + was 2.3 × 10 −2 m which agrees with the kinetically determined K A for Na + . The results are consistent with Na + binding to thrombin causing a conformational change in the active site. It is concluded that human α-thrombin is a monovalent cation-activated enzyme.

Activation of factor X: Kinetic properties of the reaction☆

Abstract A continuous spectrophotometric assay for the conversion of Factor X to Factor Xa has been developed. The assay has been used to determine the Km of Factor X in the reaction catalyzed by coagulant protein from Russells viper venom and by the intrinsic pathway of coagulation. Linear double reciprocal plots, indicative of Michaelis-Menten kinetics, are obtained with both reactions. Inosithin decreases the Km for Factor X in the reaction catalyzed by the intrinsic pathway but has no effect on the Vmax.

Purification and activation of human factor X: Cooperative effect of Ca++ on the activation reaction☆

Abstract Human Factor X has been purified 6000 fold by a combination of DEAE-Sephadex, heparin-Sepharose and hydroxylapatite chromatography. The preparation is virtually homogeneous with a monomeric molecular weight of 75,000 as judged by polyacrylamide gel electrophoresis. The activation of Factor X by the coagulant protein of Russells viper venom has been investigated utilizing Bzue5f8Ileue5f8Gluue5f8Glyue5f8Argue5f8pue5f8nitroanilide (S-2222). Human Factor Xa hydrolyzes S-2222 with a Km of 0.6 mM. The coagulant protein has a Km for human Factor X of 0.01 μM, 10-fold lower than that of bovine Factor X. Ca ++ increases the Vmax of the activation reaction and exhibits a cooperative effect. Hill plots demonstrate that there are at least three cooperative sites for Ca ++ binding. These studies indicate that Ca ++ binding to human Factor X induces a conformation change which increases the rate of the cleavage of the peptide bond required for activation.

Characterization of human platelet glutathione reductase

Glutathione reductase (NAD(P)h:oxidized glutathione oxidoreductase, EC 1.6.4.2) has been purified 1000-fold from the cytoplasmic fraction of human platelets. Salts, including the heretofore unreported effect of sodium citrate, activate the NADPH-dependent reduction of oxidized glutathione. Sodium citrate and monovalent salt activation appears to involve multiple sites having different binding affinities. At sub-saturating sodium phosphate, non-linear double reciprocal plots indicative of substrate activation by oxidized glutathione were observed. Initial velocity double reciprocal plots at sub-saturating and saturating concentrations of phosphate generate a family of converging lines. NADP+ is a partial inhibitor, indicating that the reduction of oxidized glutathione can proceed by more than one pathway. FMN, FAD, and riboflavin inhibit platelet glutathione reductase by influencing only the V while nitrofurantoin inhibition is associated with an increase Koxidized glutathione and a decreased V.

Use of Size-Exclusion High-Performance Liquid Chromatography for the Analysis of the Activation of Prothrombin,

Abstract Prothrombin is a single-chain protein ( M r 72,000) which can be converted to the two-chain coagulation enzyme thrombin ( M r 37,000) by a protein present in the venom of Echis carinatus . During the course of this reaction, several intermediates and products are produced. We have found that size-exclusion high-performance liquid chromatography is a useful method for identifying the various intermediates as well as for determining the rates at which they are produced. The intermediates were identified by comparison with either authentic proteins or sodium dodecyl sulfate-polyacrylamide gel electrophoresis of duplicate samples. The separation of prothrombin, thrombin, and the other products was accomplished in less than 18 min using a TSK Type SW 3000 column equilibrated with 0.1 m Tris buffer, pH 7.2, containing 0.15 m NaCl. Estimates of the molecular weights of the products determined from calibration curves did not necessarily agree with actual values. Meizothrombin, the two-chain active form of prothrombin, has a longer elution time than prothrombin, although both molecules have the same molecular weight. Ca(II) increased the elution time of prothrombin and Fragment 1. The rate of disappearance of prothrombin as determined by peak-height analysis agreed with the rate of formation of active enzyme as determined by active site titration. Thus, high-performance liquid chromatography is a rapid analytical tool for the study of the proteolytic modification of proteins.

Inhibition of human coagulation factor Xa by thrombin substrates

Abstract The thrombin specific chromogenic substrates, Phe-Pipecolyl-Arg-p-nitroanilide (S-2238) and benzoyl-Phe-Val-Arg-p-nitroanilide (S-2160) are inhibitors of factor Xa amidolytic and proteolytic activity. The inhibition is of the partial noncompetitive type in that no change in the Km for the hydrolyzed substrate was observed and some factor Xa activity remains even at infinite inhibitor concentration. High performance liquid chromatographic analysis indicated that the inhibitors were essentially free of either p-nitroaniline or free peptide contamination. Neither p-nitroaniline nor acid hydrolyzed inhibitors were capable of inhibiting factor Xa demonstrating that a minimal peptide structure is necessary for inhibition. Our results indicate that factor Xa contains a regulatory site which, when occupied by certain peptides, causes a decrease in the rate of catalysis.

Characterization of a membrane-associated NADH-dependent cytochrome C reductase of human platelets

Abstract Platelet membranes isolated by the glycerol lysis technique contain a NADH-dependent reductase which reduces cytochrome c. The K NADH of the membrane associated enzyme was 1.3 μM and the Vmax of the reaction was 4600 nmoles cytochrome c reduced/min/mg protein. Superoxide dismutase had no affect on the reaction indicating that reduction was independent of superoxide formation. The reductase was inhibited by the drugs quinacrine HC1, quercetin and tetracaine HC1 and also by PCMBS(p-chloromercuribenzoate sulfonate). Aggregation in citrated plasma induced by ADP, collagen and the ionophore A23187 were also inhibited by quinacrine HC1, quercetin and tetracaine HC1. It is possible that the inhibition of both the reductase activity and aggregation by these compounds reflects a role for the membrane-associated reductase in one of the processes which leads to the aggregation of platelets.