DEPLETION OF PHOSPHATE IN ACTIVE MUSCLE-FIBERS PROBES ACTOMYOSIN STATES WITHIN THE POWERSTROKE

Variation in the concentration of orthophosphate (P-i) in actively con tracting, chemically skinned muscle fibers has proved to be a useful p robe of actomyosin interaction. Previous studies have shown that isome tric tension (P-o decreases linearly in the logarithm of [P-i] for [P- i] greater than or equal to 200 mu M. This result can be explained in terms of cross-bridge models in which the release of P-i is involved i n the transition from a weakly bound, low-force actin.myosin.ADP.P-i s tate to a strongly bound, high-force, actin.myosin.ADP state. The 200 mu M minimum [P-i] examined results from an inability to buffer the in trafiber, diffusive buildup of P-i resulting from the fiber ATPase. In the present study, we overcome this limitation by employing the enzym e purine nucleoside phosphorylase with substrate 7-methylguanosine to reduce the calculated internal [P-i] in contracting rabbit psoas fiber s to <5 mu M. At 10 degrees C we find that P-o continues to increase a s the [P-i] decreases for [P-i] greater than or equal to 100 mu M. Bel ow this [P-i], P-o is approximately constant. These results indicate t hat the free energy drop in the cross-bridge power-stroke is similar t o 9kT. This value is shown to be consistent with observations of muscl e efficiency at physiological temperatures.