Addition of phosphate to active muscle fibers probes actomyosin states within the powerstroke

Abstract

We have measured the effect of phosphate (P_i) on the tension and maximum shortening velocity of permeable rabbit psoas fibers. Work in a number of laboratories has established that addition of phosphate (0–25 mM) to active muscle fibers at physiological MgATP concentrations decreases isometric tension with little effect on the maximum shortening velocity. Here we extend these results to a wider range of P_i concentrations and to low MgATP concentrations. Low levels of P_i (approx. 150 μM – 200 μM) were obtained by using sucrose phosphorylase and sucrose to reduce contaminating P_i in the solutions used to activate the fiber, and high levels (52–73 mM) were obtained by replacing acetate with P_i as the principal anion. In an activating solution containing either 50 μM or 4 mM MgATP, pH 6.2 or 7.0, isometric tension declines linearly with the logarithm of P_i concentration. Although the isometric tension decreases with increasing concentrations of H⁺ or MgATP, the slope of relative isometric tension as a function of log[P_i] is the same at the two values of pH and [MgATP]. At pH 7 and 4 mM MgATP, the velocity of contraction increased slightly as P_i increased from 0.2 to 52 mM. At 50 μM MgATP the velocity decreased slightly as P_i increased from 10 to 52 mM. These results are discussed in terms of models of cross-bridge energetics. The observation that force declines linearly with the logarithm of [P_i] is compatible with models in which a major force producing state occurs subsequent to P_i release. The inhibition of shortening velocity by P_i at low concentration of MgATP can be explained by a competition between MgATP and P_i at the end of the cross-bridge powerstroke.