Abstract
Variation in the concentration of orthophosphate (P i) in actively contracting, chemically skinned muscle fibers has proved to be a useful probe of actomyosin interaction. Previous studies have shown that isometric tension (P o) decreases linearly in the logarithm of [P i] for [P i] ≥ 200 μM. This result can be explained in terms of cross-bridge models in which the release of P i is involved in the transition from a weakly bound, low-force actin · myosin · ADP · P i state to a strongly bound, high-force, actin · myosin · ADP state. The 200 μM minimum [P i] examined results from an inability to buffer the intrafiber, diffusive buildup of P i resulting from the fiber ATPase. In the present study, we overcome this limitation by employing the enzyme purine nucleoside phosphorylase with substrate 7-methylguanosine to reduce the calculated internal [P i] in contracting rabbit psoas fibers to <5 μM. At 10°C we find that P o continues to increase as the [P i] decreases for [P i] ≥ 100 μM. Below this [P i], P o is approximately constant. These results indicate that the free energy drop in the cross-bridge powerstroke is ∼9 kT. This value is shown to be consistent with observations of muscle efficiency at physiological temperatures.
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