The activity of creatine kinase in frog skeletal muscle studied by saturation-transfer nuclear magnetic resonance

Abstract

The activity of creatine kinase in intact anaerobic frog muscle at 4.degree. C at rest, and during contraction, was investigated by using saturation-transfer 31P NMR. At rest, the measured forward (phosphocreatine to ATP) reaction flux was 1.7 .times. 10-3 M .cntdot. s-1, and the backward flux was 1.2 .times. 10-3 M .cntdot. s-1. The large magnitude of both fluxes showes that creatine kinase is active in resting muscle, so the observed constancy of [phosphocreatine] demonstrates that the enzyme and its substrates are at equilibrium. The apparent discrepancy between the fluxes must arise largely from an underestimation of the backward flux, resulting from interaction of ATP with other systems, e.g., via adenylate kinase. As an estimate of both fluxes 1.6 .times. 10-3 M .cntdot. s-1 was adopted. During contraction when the creatine kinase reaction is no longer at equilibrium, the net rate of phosphocreatine breakdown, estimated directly from the change in area of the inorganic phosphate peak, was 0.75 .times. 10-3 M .cntdot. s-1. Saturation transfer indicates that the forward reaction flux remains at approx. 1.6 .times. 10-3 M .cntdot. s-1, and the backward flux decreases to about 0.85 .times. 10-3 M .cntdot. s-1. The activity of creatine kinase during contraction is large enough to account for the observation that, during contraction, the concentration of ATP falls by less than 2-3%. The reaction catalyzed by creatine kinase is driven forward during contraction by the large relative increase in the concentration of free ADP, which is more than doubled. The observation that the forward flux does not increase during contraction, and the backward flux decreases can most simply be explained on the basis of competition of reactants for a limited amount of enzyme.