Correlation between Myofibrillar Biochemistry and Muscle Fiber Mechanics using Rabbit Psoas Muscle Preparations Indicates that Phase 2 of Step Analysis Represents the Cross-Bridge Detachment Step

Abstract

Our goal is to correlate results obtained from myofibrillar suspensions and muscle fibers. For myofibrils, tryptophan fluorescence with stopped-flow apparatus was used; for fibers, tension transients with small amplitude sinusoidal length perturbations were used. Experiments were performed in identical solutions with 0.2M ionic strength at pH 7.00. The concentration of MgATP was varied to detect kinetic constants of the ATP binding step 1 (K1: dissociation constant), the cross-bridge detachment step 2 (k2, k-2: rate constants), and the ATP cleavage step 3 (k3, k-3). By following the fast rate constant at 20°C, we found in myofibrils: k2/K1=1.0∈μM−1s−1, K1=0.3mM, k2=300s−1, and k-2≈0; in fibers: k2/K1=0.23∈μM−1s−1, K1=1.58mM, k2=363s−1, k-2=180s−1. From these results we conclude that (1) ATP binding is ∼5X stronger in myofibrils than in fibers, (2) cross-bridge detachment rate is just about the same, and (3) its reversal step is almost absent in myofibrils, but it is finite in fibers. Consequently, we found a good agreement in the results obtained from myofibrils and fibers, indicating that phase 2 of tension transients from step analysis in fibers (Huxley and Simmons, 1971) represents the cross-bridge detachment step. We also studied actin-myosin cross-linked myofibrils and found no difference, indicating that cross-linking does not significantly modify steps 1-3 kinetics. We further studied the Pi effect in myofibrils, and found that Pi is a competitive inhibitor of MgATP with the inhibitory dissociation constant of 7-8mM. To deduce the kinetic constants of the ATP cleavage step, we measured the slower rate constant in fluorescence in myofibrils and found that k3+k-3=10.7s−1 at 4°C. From the Pi burst experiments using radioactive ATP, we found that K3=6.1 at 4°C. From these, k3=9.2s−1 and k-3=1.50s−1 were deduced.