Studies on the Actin Activation of Myosin Subfragment‐1 Isoenzymes and the Role of Myosin Light Chains

The actin‐activated ATPase activity of myosin subfragment 1 (S‐1) isoenzymes, separated on the basis of their alkali light chain (A1 and A2) content, has been analysed under a number of different conditions. Previous experiments on the effects of increasing actin concentrations on the ATPase have shown that the maximum turnover rate of ATP by S‐1 (A2) was about twice that of S‐1 (A1), and the K m for actin was also considerably larger for this isoenzyme. Here we show that these kinetic differences are maintained when regulated actin (actin + tropomyosin + troponin) is used in place of purified F‐actin. However, while increasing the ionic strength of the solution from 6 mM to 46 mM KCl has little effect on V for S‐1 (A2), the corresponding value for S‐1 (A1) increases to approximately equal that of S‐1 (A2), suggesting that the two isoenzymes are controlled by the same rate‐limiting process in the steady state under these conditions. The value of K m also increases with increasing ionic strength for the two isoenzymes, but that for S‐1 (A1) increases more rapidly and approaches that for S‐1 (A2). These experiments were carried out at at fixed S‐1 concentration, but the maximum rate of actin activation of the S‐1 ATPase can also be obtained from experiments where the actin concentration is fixed and the S‐1 concentration varied. Under these conditions in 6 mM KCl, the maximum rate of ATP hydrolysis expressed per mole of actin at infinite S‐1 concentration is the same for both S‐1 (A1) and S‐1 (A2), and this value numerically equals the maximum rate of hydrolysis of ATP by S‐1 (A2) expressed at infinite actin concentration. These results suggest that the rate‐limiting step in the ATPase cycle for S‐1 (A2) is the release of products from an actin · S‐1 · ADP · P i complex, while for S‐1 (A1), where the value of V at infinite actin concentration is considerably lower, a different process is rate limiting, and this process may occur before actin reassociation with the S‐1 · ADP · p i complex, using the Lymn‐Taylor kinetic model. Thus increasing the ionic strength appears to change the rate‐limiting process in the steady‐state hydrolysis of ATP by actoS‐1 (A1). These experiments are discussed in terms of the role of the alkali light chains in the myosin ATPase and muscle contraction.