A Comparison of Single Myofibrils and Single Muscle Fibers from Rabbit Psoas in Terms of MgATP Binding and Cross-Bridge Detachment Steps Studied with Sinusoidal Length Perturbations

Abstract

Single myofibrils of 40-70 μm in length and 2-4 μm in diameter were isolated from rabbit psoas muscle bundles, and cross-bridge kinetics were studied by small amplitude (0.2%) length perturbations at 15 frequencies (1-250Hz). The experiments were performed at 15°C in the presence of 0.05-10 mM MgATP, 4 mM KH2PO4, 4 mM K2HPO4, 15 mM Na2CP (creatine phosphate), 200 mM ionic strength with KAc (acetate), pCa 4.55, and pH 7.0. Two exponential processes B and C were resolved in tension transients. Their apparent rate constants (2πb and 2πc; b < c) increased ([MgATP]=0.05-1 mM) and saturated (≥1 mM) with an increase in the MgATP concentration. However, they were close together (1.4x) at [Pi]≥4 mM, hence they were combined to deduce the accurate estimate of the kinetic constants: their sum and product were analyzed as functions of [MgATP]. These analyses yielded K1=2.91 ± 0.31 mM−1, k2=288±36 s−1, and k-2=10±21 s−1 (±95% confidence limit, N=13). These results are consistent with the cross-bridge model: AM+ATP <-> (step 1) AM.ATP <-> (step 2) A+M.ATP. These kinetic constants compare to those observed in single fibers (K1=2.35±0.31 mM−1, k2=243±22 s−1, and k-2=6±14 s−1; N=8) under the same experimental conditions and analysis methods. These values are respectively not significantly different from those of myofibrils, indicating that the same kinetic constants can be deduced from myofibril and muscle fiber studies in terms of ATP binding and cross-bridge detachments steps. The fact that in myofibrils K1 is 1.24x of that of fibers, may be explained by a small concentration gradient of ATP, ADP and/or Pi in single fibers.