Cross‐hybridization of light chains of cardiac myosin isozymes: Atrial and ventricular myosins

It was shown earlier that an increase in temperature in the absence of divalent cations was conducive to light chain dissociation [ I], and that the affinity of myosin for divalent cations decreases at lower pH values, i.e., pH 6.5 [2], thus facilitating the release of light chains [2,3]. High salt concentration promotes light chain dissociation, especially light chain LCi, either in the form of NH4C1 [4] or LiCl [5]. Following dissociation of the myosin oligomer, reassociation occured with a decrease in temperature (4’C), additional excess light chains, and the presence of divalent cations [5]. In the studies of Wagner and Weeds [4] and Okamoto and Yagi [S], the myosin oligomer was both dissociated and reassociated in the presence of excess light chains, either native or foreign. Thus, for maximum dissociation of the myosin oligomer and reassociation of myosin light chains with isozymal heavy chains without denaturing myosin, i.e., losing actin + Mg2+ -stimulated ATPase activity, the procedures of Higuchi et al. [l] were coupled with those of Wagner and Weeds [4]; this provided a system to study the role of light chains in regulation of ATPase activity. For these analyses the three myosin isozymes, sheep atrial and ventricular myosins, and rabbit skeletal muscle myosin light chains were used because of their obvious difference in ATPase activity [6,7], and their variance in light chain electrophoretic mobility [6,7]. These differences allowed the monitoring of cross-hybridization by assessing ATPase activity and mobility of light chains on SDS-polyacrylamide gel electrophoresis of the hybrid myosin.