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C M Jackson

Publications and source records attributed to C M Jackson.

At least 73 records · Page 4Linked to original sources
PubMed1986

The action of factor Xa on peptide p-nitroanilide substrates: substrate selectivity and examination of hydrolysis with different reaction conditions.

Kinetic parameters for the action of bovine Factor Xa (EC 3.4.21.22) on 25 commercially available peptide p-nitroanilides have been determined. The selectivity constant, kc/Km, ranges from 1.5 X 10(1) to 2 X 10(6) M-1 X s-1 for the poorest and the best substates, respectively. The best substrates for Factor Xa were identified as those with arginine in the P1 position, and glycine in the P2 position. Quantitative distinction between lysine and arginine in the P1 position and other amino acids in the P2-P4 positions of the substrate is reported from the changes in the kinetic parameters for substrates differing in only a single amino acid in these positions. Effect of NaCl and CaCl2 concentrations and temperature on the action of Factor Xa on selected substrates have been assessed. Km values for Factor Xa hydrolysis of most substrates are greater than 100 microM. Solubility of the substrates consequently restricts measurements of reaction velocities to concentrations lower than desirable for optimally determining kc. Comparison of these kinetic parameters for Factor Xa with those of thrombin (Lottenberg, R., Hall, J.A., Blinder, M., Binder, E. and Jackson, C.M. (1983) Biochim. Biophys. Acta 742,539-557) for these same substrates indicates that the greater hydrolytic efficiency of thrombin is due primarily to lower Km values.

Anilides↗
PubMed1986

Reaction of thrombins with human antithrombin III: II. Dependence of rate of inhibition on molecular form and origin of thrombin.

The rate of thrombin inhibition by AT III depends upon the molecular form (alpha, beta, gamma) and species origin of the enzyme. The following apparent second order rate constants (.1000/M.s) were established alpha human 11.24 +/- 0.8; alpha bovine 7.46 +/- 0.27; beta bovine 6.49 +/- 0.34 and gamma human thrombin 2.80 +/- 0.11, 25 degrees C, pH = 7.80, 0.01 M TRIS, 0.01 M HEPES buffer, 0.0025 M EDTA, 0.3 M NaCl, 1 mg/mL PEG 6000. Using these values, the concentration of active AT III in an unknown sample can be calculated from the measured apparent first order rate constant in moles/liter instead of relative units. In contrast to the reactions in the absence of heparin, in the presence of high affinity heparin, the differences between various forms of thrombin are more pronounced and the shape of the progress curves, as well as rates, are highly dependent on the ionic strength. In the presence of heparin, measurement of the rate of inhibition under pseudo first order conditions can be made only when the NaCl concentration is at least 0.3 M. The significance of the presented data for designing a functional assay of AT III is discussed.

Animals↗
PubMed1986

Preparation and properties of derivatives of bovine factor X and factor Xa from which the gamma-carboxyglutamic acid containing domain has been removed.

Limited proteolysis of bovine blood coagulation Factor X by chymotrypsin produces a derivative in which the light chain is cleaved between Tyr 44 and Lys 45. Two peptide products, residues 1-44 of the Factor X light chain and a modified zymogen, Factor X(-GD) have been isolated and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, elution behavior on anion-exchange chromatography, amino acid composition, and by partial amino acid sequence determination. Factor X(-GD) no longer contains the 12 gamma-carboxyglutamic acid residues of the native zymogen and thus serves as a model for investigation of the properties conferred on Factor X by the presence of gamma-carboxyglutamic acid. Cleavage of Factor X at Tyr 44 by chymotrypsin is inhibited by Ca2+ and Mg2+ ions. Factor X(-GD) is activated by the coagulation factor activator of Vipera russellii venom, but at less than 1% of the rate of activation of native Factor X. The susceptibility of Tyr 44 to chymotryptic cleavage implies that this residue is on the surface of the light chain of Factor X. Factor Xa(-GD) is indistinguishable from native Factor Xa in its activity on Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide, on prothrombin alone, and on prothrombin plus Factor Va. In the presence of phospholipid the rate of prothrombin activation catalyzed by Factor Xa(-GD) is the same as in the absence of phospholipid.

1-Carboxyglutamic Acid↗
PubMed1986

Localization of the structural difference between bovine blood coagulation factors X1 and X2 to tyrosine 18 in the activation peptide.

Bovine Factor X is isolated in two chromatographically separable forms, Factor X1 and Factor X2. Whereas only a single form of Factor Xa, the active protease, exists, the activation peptides also exist as two chromatographically distinct species. These peptides have been shown to differ at a tyrosyl residue by ultraviolet spectrophotometry, and in their composition after alkaline hydrolysis. On the basis of the spectral properties, and elution position of the modified tyrosine on Dowex 1 columns and on an amino acid analyzer, it has been concluded that Factor X2 contains a tyrosyl-O-SO4 residue at position 18 in the activation peptide whereas Factor X1 contains only tyrosine. Alternative explanations such as differences in carbohydrate composition, differences in phosphate content, or differences in the number of gamma-carboxyglutamic acid residues were demonstrated to be unrelated to the difference in chromatographic behavior between bovine Factors X1 and X2.

Amino Acids↗
PubMed1985

A kinetic model describing the interaction of bovine prothrombin fragment 1 with calcium ions.

A kinetic model is derived for the interaction of bovine prothrombin fragment 1 with calcium ions. The model requires binding of a minimum of two calcium ions for induction of the observed biphasic fluorescence decrease as a function of time. The model is shown to be consistent with experimental kinetic and equilibrium data by fitting theoretical curves for the biphasic fluorescence change to the data through exact solution of the nonlinear differential rate equations derived from the model. The rate constants for the binding of these two required calcium ions are calculated from the solutions as best fit parameters. The thermodynamic equilibrium constants, K1 and K2, for the binding of these two calcium ions are calculated from ratios of the forward and reverse rate constants as 0.6 X 10(4) and 5.4 X 10(4), respectively. Thus, the model correctly predicts positively cooperative calcium ion binding for at least the two calcium ions required to induce fluorescence quenching.

Animals↗
PubMed1984

Comparative protective actions of gonadotrophins and testosterone against the antispermatogenic action of ethane dimethanesulphonate.

After a single dose of ethane dimethanesulphonate (EDS) (75 mg/kg) to rats the prolonged antispermatogenic action is due to a temporary elimination of the functional Leydig cell population. Replacement therapy with testosterone propionate (3 mg/day) maintains the spermatogenic epithelium but the EDS effect develops when hormone treatment is discontinued. In contrast, a short treatment with hCG (10-100 i.u./day) or LH (714 micrograms/day), starting before the EDS dose, permanently protects the spermatogenic epithelium. FSH treatment was completely ineffective. Although histological protection of spermatogenesis appeared complete with testosterone or hCG, effects on fertility remained but over different periods of time. Antispermatogenic and antifertility effects were produced in mice using much higher doses of EDS (5 X 250 mg/kg) but there was no protection from androgen or hCG. It is suggested that EDS binds to Leydig cells irreversibly, interfering with the action of gonadotrophin. At the dose level used the evidence suggests that the degree of reaction renders most of the Leydig cell population non-viable. A direct cytotoxic effect of the compound upon the spermatogenic epithelium might account for the inability of testosterone or hCG alone or in combination to maintain fertility at normal levels.

Animals↗
PubMed1984

Factor X.

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Amino Acid Sequence↗
PubMed1983

The action of thrombin on peptide p-nitroanilide substrates. Substrate selectivity and examination of hydrolysis under different reaction conditions.

Kinetic parameters for the action of bovine alpha-thrombin on 24 commercially available peptide p-nitroanilides have been determined. The selectivity constant, kcat/Km, ranges from 3.3 X 10(1) to 1.1 X 10(8) M-1 X S-1 for the poorest and the best substrates, respectively. The best substrates for thrombin were identified as those with arginine in the P1 position, proline or a proline homolog in the P2 position, and an apolar amino acid in the P3 position. Quantitative distinction between lysine and arginine in the P1 position and other amino acids in the P2-P4 positions of the substrate is reported from the changes in the kinetic parameters for substrates differing in only a single amino acid in these positions. Effects of NaCl, CaCl2 and poly(ethylene glycol) concentrations, pH and temperature on the action of thrombin on selected substrates have been assessed. A source of large systematic error in thrombin concentration estimates was identified as resulting from adsorption losses. These losses were eliminated by inclusion of poly(ethylene glycol) in dilution and reaction buffers.

Aniline Compounds↗
PubMed1983

Solution composition dependent variation in extinction coefficients for p-nitroaniline.

The dependence of the extinction coefficients for para-nitroaniline on solution composition has been investigated. The p-nitroaniline absorption spectrum is red-shifted with increasing ionic strength, with the consequence that the extinction coefficients at fixed wavelengths may vary significantly. The isosbestic wavelength for peptide p-nitroanilide/p-nitroaniline mixtures is similarly shifted. Poly(ethylene glycol) and bovine albumin, two additives frequently employed to eliminate enzyme loss from adsorption to cuvette and dilution vessel surfaces, also induce shifts in the p-nitroaniline spectrum. The use of a difference extinction coefficient at 381 nm, the p-nitroaniline maximum wavelength, is proposed to minimize the error resulting from solution composition dependent spectral shifts.

Aniline Compounds↗
PubMed1982

The action of thrombin on peptide p-nitroanilide substrates: hydrolysis of Tos-Gly-Pro-Arg-pNA and D-Phe-Pip-Arg-pNA by human alpha and gamma and bovine alpha and beta-thrombins.

Human and bovine alpha-thrombins (greater than 90% alpha form) with high fibrinogen clotting activities (approximately 3,000 U.S. units/mg protein) exhibit similar Michaelis menten kinetics with the p-nitroanilide tripeptide substrates Tos-Gly-Pro-arg-pNA (Chromozym-TH) and D-Phe-Pip-Arg-pNA (S-2238). The kinetic parameters at I = 0.11 M, 25 degrees C, pH 7.8 are: (Km = 4.18 +/- 0.22 and 3.61 +/- 0.15 microM; kcat = 127 +/- 8 and 100 +/- 1 s-1) for Chromozym TH and (Km = 1.33 +/- 0.07 and 1.50 +/- 0.10 microM; kcat = 91.4 +/- 1.8 and 98.0 +/- 0.5 s-1) for S-2238 for the human and bovine enzymes, respectively. Unlike the native enzyme forms, their "non-clotting" terminal degradative forms, human gamma-thrombin (approximately 5 units/mg) and bovine beta-thrombin (approximately 200 units/mg), give increased values for these parameters (km = 14.3 +/- 2.4 and 14.4 +/- 2.2 microM; kcat = 160 +/- 9 and 124 +/- 6 s-1) for Chromozym-TH; and (Km = 2.50 +/- 0.36 and 2.99 +/- 0.33 microM; kcat = 106 +/- 3 and 106 +/- 3 s-1) for S-2238. Based on these parameters, 50% degradation of human or bovine alpha-thrombins can be calculated to produce relatively small errors in the kinetic measurement of total thrombin concentrations (maximally 9% and 7% for Chromozym-TH; 7% and 3% for S-2238, respectively) if the kinetic parameters for all alpha forms are erroneously used and assays are at 150 microM substrate. This is in contrast to the large errors inherent in clotting activity measurements on thrombin mixtures. Incorporation of 1 mg/ml of polyethylene glycol 6,000 into assay solutions eliminates systematic errors otherwise caused by thrombin adsorption to surfaces and enables thrombin to be accurately assayed at concentrations less than 0.1 nM or 0.01 clotting unit/ml of alpha-thrombin.

Adsorption↗
PubMed1982

The active site of antithrombin. Release of the same proteolytically cleaved form of the inhibitor from complexes with factor IXa, factor Xa, and thrombin.

Reactions between near equimolar amounts of antithrombin and Factors IXa or Xa resulted in the formation of a free proteolytically modified, two-chain form of the inhibitor, in addition to the inactive antithrombin-protease complexes. The modified inhibitor produced by either enzyme was electrophoretically identical with that formed in the reaction with thrombin. As in the latter reaction, the formation of the modified antithrombin by Factor Xa was increased in the presence of heparin, while only small amounts were produced by Factor IXa both in the absence and presence of the polysaccharide. NH2-terminal sequence analyses of the isolated modified inhibitor formed by Factor Xa showed that a single Arg-Ser bond in the COOH-terminal end of the inhibitor had been cleaved. This cleavage site is identical with that identified in free thrombin-modified antithrombin. The purified antithrombin-Factor IXa and antithrombin-Factor Xa complexes were dissociated by ammonia or hydroxylamine into free enzyme and a modified two-chain form of the inhibitor. Electrophoresis studies and NH2-terminal sequence analyses showed that the modified antithrombin obtained from either complex was identical with that produced in free form by the two enzymes and also with the modified inhibitor that is released from the antithrombin-thrombin complex. The fact that identical results were obtained for the reactions between antithrombin and three enzymes with different specificities strongly suggests that the observed Arg-Ser cleavage site is the active site of antithrombin.

Amino Acid Sequence↗
PubMed1981

Calcium ion-protein interactions in prothrombin activation.

1. The protein concentration dependence observed in the calcium binding to fragment 1 indicates that calcium-mediated dimerization is responsible for the cooperative calcium binding behavior usually observed. "Unusual" fragment 1, which exhibits negative cooperativity (the type of binding behavior expected for ions interacting with a charged protein) at high concentration, also exhibit altered self-association behavior. 2. The calcium-induced spectral perturbations that are observed by fluorescence and ultraviolet difference spectroscopy are influenced by calcium-mediated dimerization. Similar spectral perturbations may also be induced by other divalent, trivalent, and monovalent ions, as well as changes in pH. Because this is a multi-site system, only limited interpretation of the spectral data is possible without calcium binding data. 3. Although strong side chain CD signals make estimation of fragment 1 secondary structure ambiguous, the CD data do indicate small changes in structure during calcium binding. Similar changes are observed upon addition of monovalent ions at high concentration or after lowering the pH. No coupling between changes in conformation and the cooperative calcium binding behavior has yet been observed to exist.

Binding Sites↗
PubMed1980

Blood coagulation.

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Amino Acid Sequence↗
PubMed1979

On the molecular-weight-dependence of the anticoagulant activity of heparin.

The inactivation of thrombin and factor Xa by antithrombin was determined in the presence of heparin fractions of different molecular weights and with high affinity for antithrombin. The ability to potentiate the inactivation of either coagulation factor increased with increasing length of the polysaccharide chain.

Antithrombin III↗