Search

The role of Ca 2+ in regulation of platelet actomyosin ATPase activity has been investigated. The results suggest that Ca 2+ has at least two roles in the reaction mechanism; (a) it forms a complex with ATP to form the substrate, CaATP and (b) it forms a complex with the protein to activate the enzyme. Both the substrate and free Ca 2+ bind cooperatively to the protein. The binding of free Ca 2+ stimulates the enzymic activity and causes a change in the apparent K m value. The apparent K m value for CaATP is 0.15mM in the absence of free Ca 2+ and 0.07mM in the presence of 2.5mM Ca 2+ . Thus Ca 2+ appears to act as a positive allosteric effector.

The effects of various modifiers on the ATPase activity of bovine platelet actomyosin has been studied. The order of activation by monovalent cations was NH 4 + ⪢ K + > Li + > Na + . The order of activation by divalent cations was Ca 2+ > Mn 2+ = Sr 2+ > Ba 2+ > Co 2+ > Mg 2+ > Zn 2+ . Ethylenediaminetetraacetate inhibits. Activity increased with increasing concentrations of monovalent cations, except for inhibition by increasing concentrations of NH 4 + in the presence of Ca 2+ . Adenosine triphosphatase activity was increased by low concentrations of urea and trypsin, but was unaffected by low concentrations of N -ethylmaleimide. For all enzymatic properties where direct comparisons are possible, actomyosin from platelets is unlike that from skeletal muscle, but is similar to that from smooth muscle and non-muscle sources.

The effects of Mg2+, Mn2+, Zn2+, Co2+, Ni2+, Ca2+, and Sr2+ upon the equilibrium constant (K) and formation rate constant (k1) of H-meromyosin-ADP complex were studied. The affinity constant of ADP binding decreases in the following order Mn > Mg > Zn > Ni > Co > Sr ∼- Ca. The calculated dissociation rate constant (k2) of the enzyme-ADP complex was similar to the rate of ATP hydrolysis with all divalent metals tested, except Sr2+. With Mg as predominant metal ion the following dissociation constants were obtained for the binding of various nucleoside diphosphates to H-meromyosin: ADP 1–2 × 10−6 m , UDP 2 × 10−5 m , CDP 3 × 10−5 m , IDP 10−4 m . The results are compatible with the suggestion that at 6 °C the dissociation of ADP from the active site limits the rate of ATP hydrolysis with Mg2+, Mn2+, Co2+, and Ca2+.

The kinetic properties of platelet actomyosin have been examined to understand the mode of hydrolysis of its substrate ATP. In the presence of divalent cations, ATP hydrolysis deviated from Michaelis-Menten kinetics in such a way as to indicate cooperative effects, with a sigmoidal velocity vs. substrate curve and a Hill slope of 2.4. In the absence of added divalent cations, linear Michaelis-Menten kinetics were obtained and the Hill slope reduced to 1.0. These results indicate an allosteric regulatory site on platelet actomyosin.

Reaction of myosin or H-meromyosin with 10–20 moles of N-ethylmaleimide (NEM) per mole of enzyme inhibits the Mg++, Ca++ or EDTA-moderated ATPase activity, while the affinity of the enzyme for ADP increases. Inhibition of ADP binding requires 40–200 moles of NEM per mole of enzyme, suggesting the involvement of distinct classes of SH groups in the catalytic function and in the binding of ADP to myosin. Rapid kinetic measurements in a stopped-flow apparatus indicate that small concentrations of N-ethylmaleimide (2–5 moles per mole of enzyme) which activates the Mg2+ or Ca2+-moderated ATPase activity at 25 °, and inhibits at 6 °, decrease the rate of appearance and the magnitude of the ATP- or ADP-induced difference spectrum of H-meromyosin at 288 mμ. Assuming that the rate of appearance of the difference spectrum is a measure of the rate of interaction of ADP with H-meromyosin, SH group modification decreases both the formation (k1) and dissociation (k2) rate constants of the H-meromyosin-ADP complex. The decrease in k2 is accompanied by the inhibition of Mg-moderated ATPase activity by N-ethylmaleimide at 6 °. The ultraviolet difference spectrum at 288 mμ, which accompanies the binding of ATP or ADP to H-meromyosin is abolished by N-ethylmaleimide without major interference with the substrate binding.

The effect of N -ethylmaleimide on the ATPase activity and ADP binding of tryptic H-meromyosin was studied at 6 and 23 °C temperatures. The affinity constant of H-meromyosin for ADP with Mg as activator was increased by small concentrations of N -ethylmaleimide (2.25 moles per mole of enzyme) at both temperatures, accompanied by activation of ATP hydrolysis at 25 °C and inhibition at 6 °C. With higher N -ethylmaleimide concentrations, the ATPase activity was inhibited at both temperatures, without comparable inhibition of ADP binding. Rapid kinetic analysis of the rate of development of difference spectrum after the addition of ATP or ADP to H-meromyosin indicates, that blocking of the S 1 and S 2 SH groups of H-meromyosin decreases both the formation ( k 1 ) and the dissociation ( k 2 ) rate constants of H-meromyosin substrate complex. At 6 °C, in the presence of Mg, the value of k 2 for ADP is similar to the turnover number of ATP hydrolysis, suggesting that dissociation of ADP from the active site may be the rate-limiting step of ATP hydrolysis. At 23 °C, the turnover number of Mg-moderated ATP hydrolysis is much smaller than k 2 , indicating that the rate limitation shifted so another, so far unidentified, step.