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M C Scrutton

Publications and source records attributed to M C Scrutton.

At least 73 records · Page 4Linked to original sources
PubMed1979

Factors influencing the response of human blood platelets to analogues of ADP which may act as partial agonists at the ADP receptor.

1. The prior addition of non-aggregating concentrations of the divalent cation ionophore, A-23187, causes human platelets to aggregate in response to a subsequent addition of the 2',3'-dialdehyde and 2',3'-dialcohol derivatives of ADP (oADP and or ADP). Previous studies [Pearce et al. (1978) Eur. J. Biochem. 88, 543--555] have shown that these derivatives act as partial agonists at the platelet ADP receptor inducing only the transition from discoid to globular morphology ('shape change'). A secretion response is also observed on addition of a low concentration of ionophore A-23187 prior to orADP. These responses are not observed if ionophore A-23187 is added prior to the 2',3'-dialdehyde and 2',3'-dialcohol derivatives of ATP (oATP and or ATP) and are markedly inhibited by prior addition of the ADP antagonist, adenosine 5'-[beta, gamma-methylene]triphosphate. 2. The aggregation response to oADP in the presence of ionophore A-23187 is reduced but not eliminated by addition of 3 mM EGTA when studies are performed in heparinised platelet-rich plasma. Additions of 3 mM EGTA in citrated platelet-rich plasma, or of 4 mM EDTA in either system completely inhibits this response. Inhibitors which are reported to elevate the intracellular concentration of adenosine 3':5'-monophosphate (cyclic AMP) or to prevent Ca2+ movement also inhibit the aggregation response to oADP which is observed in the presence of ionophore A-23187. 3. Prior addition of inhibitors of adenylate cyclase fails to cause an aggregation response to subsequent addition of oADP or orADP. Certain of these inhibitors enhance and prolong the shape change response to oADP or orADP but only at concentrations an order of magnitude in excess of those required to antagonise inhibition by agents such as prostaglandin E1, which act by increasing the concentration of cyclic AMP. 4. The concentration of prostaglandin E1, adenosine or papaverine required to inhibit shape change induced by oADP is one to two orders of magnitude lower than that required to inhibit shape change induced by ADP. 5. Prior addition of oADP decreases the lag phase in the response of human platelets to arachidonate while also increasing the concentration required to observe half-maximal response, and causing a decrease in the extent of the response. Prior addition of oATP also diminishes the extent of this response and increases the concentration of arachidonate required but has no effect on the lag phase. 6. The data suggest that oADP and orADP are capable only of acting as partial agonists at the ADP receptor because of a defective ability to increase cytosolic Ca2+ concentration. The defect is rectified by the presence of low concentrations of ionophore A-23187, which promotes mobilisation of Ca2+ from an intracellular store. The results do not appear consistent with the thesis that a decrease in platelet cyclic AMP is an initiating event in aggregation induced by ADP, but do support a model which implicates cyclic AMP in depletion of cytosolic Ca2+.

Adenosine Diphosphate↗
PubMed1979

Inhibitory effects of histidine and their reversal. The roles of pyruvate carboxylase and N10-formyltetrahydrofolate dehydrogenase.

1. N10-Formyltetrahydrofolate dehydrogenase was purified to homogeneity from rat liver with a specific activity of 0.7--0.8 unit/mg at 25 degrees C. The enzyme is a tetramer (Mw = 413,000) composed of four similar, if not identical, substrate addition and give the Km values as 4.5 micron [(-)-N10-formyltetrahydrofolate] and 0.92 micron (NADP+) at pH 7.0. Tetrahydrofolate acts as a potent product inhibitor [Ki = 7 micron for the (-)-isomer] which is competitive with respect to N10-formyltetrahydrofolate and non-competitive with respect to NADP+. 3. Product inhibition by NADPH could not be demonstrated. This coenzyme activates N10-formyltetrahydrofolate dehydrogenase when added at concentrations, and in a ratio with NADP+, consistent with those present in rat liver in vivo. No effect of methionine, ethionine or their S-adenosyl derivatives could be demonstrated on the activity of the enzyme. 4. Hydrolysis of N10-formyltetrahydrofolate is catalysed by rat liver N10-formyltetrahydrofolate dehydrogenase at 21% of the rate of CO2 formation based on comparison of apparent Vmax. values. The Km for (-)-N10-folate is a non-competitive inhibitor of this reaction with respect to N10-formyltetrahydrofolate, with a mean Ki of 21.5 micron for the (-)-isomer. NAD+ increases the maximal rate of N10-formyltetrahydrofolate hydrolysis without affecting the Km for this substrate and decreases inhibition by tetrahydrofolate. The activator constant for NAD+ is obtained as 0.35 mM. 5. Formiminoglutamate, a product of liver histidine metabolism which accumulates in conditions of excess histidine load, is a potent inhibitor of rat liver pyruvate carboxylase, with 50% inhibition being observed at a concentration of 2.8 mM, but has no detectable effect on the activity of rat liver cytosol phosphoenolpyruvate carboxykinase measured in the direction of oxaloacetate synthesis. We propose that the observed inhibition of pyruvate carboxylase by formiminoglutamate may account in part for the toxic effect of excess histidine.

Animals↗
PubMed1978

Interaction of human blood platelets with the 2',3'-dialdehyde and 2',3'-dialcohol derivatives of adenosine 5'-diphosphate and adenosine 5'-triphosphate.

1. The 2',3'-dialdehyde derivative of ADP (oADP) at concentrations approaching the millimolar range induces human blood platelets to undergo the transition from discoid to globular morphology (the 'shape change') but is incapable of inducing aggregation. 2. When incubated with platelets for 1 min before addition of the agonist, oADP acts as a competitive inhibitor of shape change and aggregation induced by ADP. Under these conditions secretion and hence aggregation induced by low concentrations of collagen; and secretion and hence secondary aggregation induced by adrenaline, thrombin and vasopressin are also inhibited by this analogue. In addition, oADP stimulates the rate of primary aggregation induced by adrenaline and causes partial inhibition of primary aggregation induced by thrombin or vasopressin. When longer preincubation times are employed the extent of inhibition with respect to all agonists, except for high concentrations of collagen, is increased and the competitive character of the inhibition with respect to ADP is no longer apparent. 3. Incubation of human platelets with the 2',3'-dialdehyde derivative of ATP (oATP) causes effects similar to those described for oADP except that the analogue neither induces platelet shape change, nor stimulates the rate of primary aggregation induced by adrenaline. In addition oATP fails to cause significant inhibition of platelet shape change induced by serotonin. The extent and character of inhibition caused by addition of oATP is not a function of the time of incubation. 4. The 2',3'-dialcohol derivatives of ADP and ATP and orATP) effect the aggregation properties of human blood platelets in a manner generally resembling those observed for the 2',3'-dialdehyde analogues. However, orADP is only weakly effective in causing platelet shape change and stimulating the rate of primary aggregation induced by adrenaline and does not inhibit secretion induced by adrenaline, collagen, thrombin and vasopressin. The extent of inhibition by orADP increases only slightly with increased time of incubation. 5. The data suggest that oADP acts as a partial agonist, and oATP as an antagonist, at the platelet ADP receptor, but that platelet membrane stabilisation also results from interaction with these dialdehyde analogues. Such membrane stabilisation does not complicate the interaction of platelets with orADP, which appears to act as a classical antagonist for the ADP receptor.

Adenosine Diphosphate↗
PubMed1978

Pyruvate carboxylase from a thermophilic Bacillus. Studies on the specificity of activation by acyl derivatives of coenzyme A and on the properties of catalysis in the absence of activator.

1. Oxaloacetate synthesis catalysed by pyruvate carboxylase from a thermophilic Bacillus in the absence of acetyl-CoA required addition of high concentrations of pyruvate, MgATP(2-) and HCO(3) (-), and at 45 degrees C occurred at a maximum rate approx. 20% of that in the presence of a saturating concentration of acetyl-CoA. The apparent K(m) for HCO(3) (-) at pH7.8 was 400mm without acetyl-CoA, and 16mm with a saturating activator concentration. The relationship between reciprocal initial rate and reciprocal MgATP(2-) concentration was non-linear (convex-down) in the absence of acetyl-CoA, but the extent of deviation decreased as the activator concentration was increased. The relationship between reciprocal initial rate and reciprocal pyruvate concentration was non-linear (convex-down) in the presence or absence of acetyl-CoA. 2. The optimum pH for catalysis of oxaloacetate synthesis was similar in the presence or absence of acetyl-CoA. The variation with pH of apparent K(m) for HCO(3) (-) implicated residue(s) with pK(a) 8.6 in catalysis of the activator-independent oxaloacetate synthesis. 3. Linear Arrhenius and van't Hoff plots were observed for the temperature-dependence of oxaloacetate synthesis in the absence of acetyl-CoA over the range 25-55 degrees C. E(a) (activation energy) was 56.3kJ/mol and DeltaH(double dagger) (HCO(3) (-)) (enthalpy of activation) was -38.6kJ/mol. In the presence of acetyl-CoA, biphasic Arrhenius and van't Hoff plots are observed with a change of slope at 30 degrees C in each case. E(a) was 43.7 and 106.3kJ/mol above and below 30 degrees C respectively. 4. Incubation of Bacillus pyruvate carboxylase with trinitrobenzenesulphonate caused specific inactivation of acetyl-CoA-dependent catalytic activity associated with the incorporation of 1.3+/-0.2 trinitrophenyl residues per subunit. Activator-independent catalysis and regulatory inhibition by l-aspartate were unaffected. The rate of inactivation of acetyl-CoA-dependent catalysis by trinitrobenzenesulphonate was specifically decreased by addition of acetyl-CoA and other acetyl-CoA and other acyl-CoA species, but complete protection was not obtained. 5. All alkylacyl derivatives of CoA tested activated Bacillus pyruvate carboxylase; acetyl-CoA was the most effective. The apparent K(a) exhibited a biphasic relationship with acyl-chain length for the straight-chain homologues. Certain long-chain acyl-CoA species showed additional activation at a high concentration. Weak activation occurred on addition of CoA or adenosine 3',5'-bisphosphate, but carboxyacyl-CoA species and derivatives containing a modified phosphoadenosyl group were inhibitory. Thioesters of CoA with non-carboxylic acids, e.g. methanesulphonyl-CoA, serve as activators of the thermophilic Bacillus and Saccharomyces cerevisiae pyruvate carboxylases, but as inhibitors of pyruvate carboxylases obtained from chicken and rat liver. 6. alpha-Oxoglutarate mimics the effect of l-aspartate as a regulatory inhibitor of the pyruvate carboxylases from both the thermophilic Bacillus and Saccharomyces cerevisiae. l-Glutamate was ineffective in both cases.

Acetyl Coenzyme A↗
PubMed1978

Partial purification and some properties of pyruvate carboxylase from the flight muscle of the locust (Schistocerca gregaria).

A procedure is described for the partial purification of pyruvate carboxylase (pyruvate:CO2 ligase (ADP-forming), EC 6.4.1.1) from the flight muscle of the locust (Schistocerca gregaria). Characterisation of the kinetic properties of this enzyme indicates that it is activated by acetyl-CoA, is insensitive to inhibition by di- and tricarboxylic acids and exhibits an apparent Km for HCO3-(16 mM) which differs by an order of magnitude from that observed for other pyruvate carboxylases. It is suggested that activation of this locust flight muscle pyruvate carboxylase during the rest leads to flight transition may result from increases in the concentrations of pyruvate and HCO3- under these conditions.

Flight, Animal↗