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

Abstract

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 (Al), and the K , 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 (Al) 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 , also increases with increasing ionic strength for the two isoenzymes, but that for S-1 (Al) 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 (Al) 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 . Pi complex, while for S-1 (Al), 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 . Pi 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 (Al). These experiments are discussed in terms of the role of the alkali light chains in the myosin ATPase and muscle contraction.

Functional Effects of the Hypertrophic Cardiomyopathy R403Q Mutation Are Different in an α- or β-Myosin Heavy Chain Backbone

“…S1). The ϳ6-fold higher V max values for the S1 subfragments compared with myosin have been found for many striated muscle myosin IIs, including fast skeletal myosin (21) and slow/cardiac myosin (22). The increase in activity is probably FIGURE 6.…”

Proton Nuclear‐Magnetic‐Resonance Spectroscopy of Myosin Subfragment 1 Isoenzymes

“…The only reported differences between the two S1 isoenzymes have been in their actin-activated MgATPase activities [20,21,401, although these have been shown to disappear as the ionic strength is raised [43]. The structural changes noted above were not, however, altered as the ionic strength was raised to physiological levels.…”

Mechanoenzymatic Characterization of Human Myosin Vb

“…However, there are the regions in the motor domain of myosin Vb whose sequences are quite different from that of myosin Va. Among them, of interest is the loop 2 region that has been suggested to influence the actin binding property of myosin (43)(44)(45)(46)(47) since the K actin of myosin Vb is significantly higher than that of myosin Va. Figure 12 shows the amino acid sequence comparison between human myosin Va and myosin Vb in the motor domain. The loop 2 sequence of myosin Vb is unique and quite different from that of myosin Va.…”