关键词：
Cross-bridge
Kinetics
ATP effect
Sinusoidal analysis
Stopped flow
摘要：
Our goal is to correlate kinetic constants obtained from fluorescence studies of myofibril suspension with those from mechanical studies of skinned muscle fibers from rabbit psoas. In myofibril studies, the stopped-flow technique with tryptophan fluorescence was used;in muscle fiber studies, tension transients with small amplitude sinusoidal length perturbations were used. All experiments were performed using the equivalent solution conditions (200 mM ionic strength, pH 7.00) at 10A degrees C. The concentration of MgATP was varied to characterize kinetic constants of the ATP binding step 1 (K (1): dissociation constant), the binding induced cross-bridge detachment step 2 (k (2), k (-2): rate constants), and the ATP cleavage step 3 (k (3), k (-3)). In myofibrils we found that K (1) = 0.52 +/- A 0.08 mM (+/- 95% confidence limits), k (2) = 242 +/- A 24 s(-1), and k (-2) a parts per thousand 0;in muscle fibers, K (1) = 0.46 +/- A 0.06 mM, k (2) = 286 +/- A 32 s(-1), and k (-2) = 57 +/- A 21 s(-1). From these results, we conclude that myofibrils and muscle fibers exhibit nearly equal ATP binding step, and nearly equal ATP binding induced cross-bridge detachment step. Consequently, there is a good correlation between process C (phase 2 of step analysis) and the cross-bridge detachment step. The reverse detachment step is finite in fibers, but almost absent in myofibrils. We further studied partially cross-linked myofibrils and found little change in steps 2 and 3, indicating that cross-linking does not affect these steps. However, we found that K (1) is 2.5x of native myofibrils, indicating that MgATP binding is weakened by the presence of the extra load. We further studied the phosphate (Pi) effect in myofibrils, and found that Pi is a competitive inhibitor of MgATP, with the inhibitory dissociation constant of similar to 9 mM. Similar results were also deduced from fiber studies. To characterize the ATP cleavage step in myofibrils, we measured the slow rate constant in
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