CROSS-BRIDGE CYCLING AT REST AND DURING ACTIVATION - TURNOVER OF MYOSIN-BOUND ADP IN PERMEABILIZED SMOOTH-MUSCLE

Single turnover experiments were performed on myosin-bound ADP by meas uring the time course of incorporation of [H-3]ADP following rapid for mation of [H-3]ATP by photolysis of caged [H-3]ATP. Permeabilized rabb it portal veins were incubated in a solution at 20 degrees C with 1 mM MgATP, 20 mM phosphocreatine, 1 mg/ml creatine phosphokinase, and con taining [C-14]ATP and high specific activity caged [H-3]ATP. At variab le times following a UV flash, the muscle was frozen, nucleotides were extracted, and the ratio H-3:C-14 in ADP was compared to that in ATP. At rest, the exchange of bound ADP occurred with a rate constant of 0 .004 s(-1). When the myosin light chain was about 80% thiophosphorylat ed, and the muscle was generating maximum isometric force, there appea red a fast phase of ADP exchange (44% of the total) which had a rate c onstant of 0.2 s(-1). The change in rate of ADP exchange on myosin is sufficient to explain the measured increase in ATPase activity upon th iophosphorylation of the myosin light chain. A simple analysis of the data suggests that there is a 50-fold increase in the cycling rate of cross-bridges in the muscle upon phosphorylation under isometric condi tions. The fraction of ADP exchanged at 10 s following photolytic rele ase of [H-3]ATP was found to be approximately linearly related to the degree of thiophosphorylation of the myosin light chain. This supports the idea that phosphorylation of the light chain causes the transitio n of myosin from the resting (slow ATPase) cycle into the activated (f ast ATPase) cycle, and that the fraction of myosin in the fast cycle i s directly determined by the degree of light chain phosphorylation. Th e data are also consistent with the cooperativity model described prev iously by Vyas ct al. (Vyas, T. B., Mooers, S. U., Narayan, S. R., Wit herell, J. C., Siegman, M. J., and Butler, T. M. (1992) Am. J. Physiol . 263, C210-C219).