Effect of an ADP analog on isometric force and ATPase activity of active muscle fibers

Abstract

The role played by ADP in modulating cross-bridge function has been difficult to study, because it is hard to buffer ADP concentration in skinned muscle preparations. To solve this, we used an analog of ADP, spin-labeled ADP (SL-ADP). SL-ADP binds tightly to myosin but is a very poor substrate for creatine kinase or pyruvate kinase. Thus ATP can be regenerated, allowing well-defined concentrations of both ATP and SL-ADP. We measured isometric ATPase rate and isometric tension as a function of both [SL-ADP], 0.1–2 mM, and [ATP], 0.05–0.5 mM, in skinned rabbit psoas muscle, simulating fresh or fatigued states. Saturating levels of SL-ADP increased isometric tension (by P′), the absolute value of P′ being nearly constant, ∼0.04 N/mm 2 , in variable ATP levels, pH 7. Tension decreased (50–60%) at pH 6, but upon addition of SL-ADP, P′ was still ∼0.04 N/mm 2 . The ATPase was inhibited competitively by SL-ADP with an inhibition constant, K i , of ∼240 and 280 μM at pH 7 and 6, respectively. Isometric force and ATPase activity could both be fit by a simple model of cross-bridge kinetics.