INHIBITION OF ATP BINDING TO MYOFIBRILS AND ACTO-MYOSIN SUBFRAGMENT-1BY CAGED-ATP

The inhibitory effect of P-3-[1-(2-nitrophenyl)ethyl] adenosine 5'-tri phosphate (caged ATP) on the binding of Mg2+-ATP to myofibrils was inv estigated. The most sensitive method was found to be the monitoring of single turnovers of [gamma_P-32] ATP hydrolysis using the quench flow technique. The method was tested using ADP, which was found to have a n inhibition constant of 145 mu M, in agreement with previous reports. Caged ATP behaved as a simple competitive inhibitor of ATP binding wi th an inhibition constant of 1.6 mM. The inhibitory effect of these li gands on the binding of ATP to acto-myosin subfragement 1 was investig ated using the same method. The inhibition constants of caged ATP and ADP were found to be 0.35 mM and 50 mu M, respectively. This inhibitor y effect of caged ATP on ATP binding accounts for the lower rate of AT P binding to fibers, deduced from caged ATP [(0.5-1) X 10(6) M(-1) s(- 1)], than that reported for acto-S1 (3.5 x 10(6) M(-1) s(-1)) [Goldman , Y. E., Hibberd, M. G., and Trentham, D. R. (1984) J. Physiol. (Londo n) 354, 577].