Biphasic kinetics of ATP hydrolysis by calcium-dependent ATPase of the sarcoplasmic reticulum of skeletal muscle. Evidence for a nucleoside triphosphate effector site.

The calcium ATPase of sarcoplasmic reticulum, which pumps calcium ion from the cytosol into the reticular compartment, does not show a simple hyperbolic dependence of calcium ATPase rate on the concentration of substrate MgATP. Early workers observed that ATPase activity rose and began to plateau with MgATP concentrations from 0.5 to 10 w, but then continued to rise with MgATP concentrations into the millimolar range. The cause of the biphasic activityMgATP dependence, however, has remained obscure. In the present work we show that variations in nucleotide substrate, metal ion, pH and temperature, partial inactivation by Nethylmaleimide, and product inhibition by MgADP give no observable differential effect on the low MgATP (2 to 100 w) as opposed to the high MgATP (0.5 to 4 mu) region of the calcium ATPase steady-state rate curve. In the presence of adenylylmethylene diphosphonate, however, the calcium ATPase shows a normal hyperbolic dependence of velocity on MgATP concentration. A delipidated, monomeric preparation of the ATPase gives a nonhyperbolic velocity-MgATP curve, with V and K kinetic parameters at high MgATP equal to those for the vesicular ATPase. Four general types of mechanisms give rise to nonhyperbolic rate-substrate curves: (i) heterogeneity of enzyme active sites (due to the presence of several ATPases, for example); (ii) certain complex mechanisms involving branched reaction pathways, as in hysteretic enzyme mechanisms in which a single active site undergoes a slow transition, for example; (iii) coupling between active sites on different polypeptide chains; and (iv) coupling between an effector site for nucleoside triphosphate and the active site. Our data are not consistent with the first, second, and third causes, but indicate that the calcium ATPase has an effector site for nucleoside triphosphates which can regulate active site saturation and turnover of MgATP. We do not rule out the presence of higher aggregates (dimers, tetramers) of the sarcoplasmic reticulum ATPase, nor of slow transitions; however, they are not the cause of the biphasic dependence of steady state ATPase rate on MgATP concentration.