Steady-state kinetic studies on the actin activation of skeletal muscle heavy meromyosin subfragments: Effects of skeletal, smooth and non-muscle tropomyosins

Abstract

The addition of either smooth muscle or brain tropomyosin to skeletal muscle actoheavy meromyosin (HMM) or acto-myosin subfragment-1 (SF1) produces an activation of the actin-activated ATPase activity up to 100%. This contrasts with the opposite, inhibitory effect produced by skeletal muscle tropomyosin. The degree of activation or inhibition depends on the ionic conditions, which influence the affinities of tropomyosin and HMM or SF1 for actin as well as on the molar ratio of actin to myosin. Enzyme kinetic analysis indicates that the inhibitory effect of skeletal muscle tropomyosin results from an approximately six- to tenfold increase in the apparent affinity ( K app) of the myosin head for the F-actin-tropomyosin complex with a concomitant six- to tenfold reduction in the maximal turnover rate ( V max). Thus, there is no direct competition of skeletal muscle tropomyosin and myosin for the same site on actin. Brain tropomyosin has an opposite effect, decreasing the apparent affinity with concomitant increase in the V max. The effect of smooth muscle tropomyosin is more complex. At high ratios of myosin to actin this tropomyosin produces the same change in the K app as skeletal muscle tropomyosin but yields a value of V max that is about twofold higher. At lower molar ratios (below about 1 to 5 myosin subfragments to actin) the activating effect of this tropomyosin remains unchanged while the apparent affinity decreases to that observed for pure F-actin. On the basis of these data as well as from experiments carried out at fixed actin and varying SF1 concentrations, it is concluded that tropomyosins act in general as allosteric un-competitive inhibitors or activators of actomyosin by increasing or reducing the co-operative activation of myosin by actin at the level of product release.