Phosphorylation and the Binding of Calcium and Magnesium to Skeletal Myosin

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Abstract
The binding of Ca2+ and Mg2+ to the isolated metal-binding L chains, i.e., those dissociable by 5,5''-dithiobis(2-nitrobenzoate), of rabbit skeletal muscle myosin is moderated by phosphorylation and is accompanied by a sizeable conformational change. As judged by circular dichroism in the region of the aromatic Cotton effects, this conformational change occurs when Ca2+ bind to the L chain in situ on the myosin head. The affinity for Ca is again changed by phosphorylation. The change in chymotryptic digestion patterns, in particular the protection of the head-rod junction in insoluble myosin, by divalent cations, has been used to obtain binding profiles. The results are consistent with the presence of a single class of independent sites, showing no cooperation. The affinity of the site for Ca2+ and Mg2+ is enhanced by 1-2 orders of magnitude when the L chain is incorporated in the myosin heads. The effect of phosphorylation on the affinity persists in these circumstances, marked for Ca and small for Mg. On phosphorylation, the Ca binding constant falls from 8 .times. 106 to 4 .times. 106 M-1 at physiological ionic strength, compared with 2.5 .times. 105 and 5 .times. 104 M-1 for the isolated L chains. The sensitivity of the proteolytic cleavage sites is affected by phosphorylation. In the absence of Ca2+ the yield of subfragment 1 at a low chymotrypsin concentration is substantially greater in dephosphorylated than phosphorylated myosin; at saturating concentrations of Ca2+, attack at the light meromyosin/heavy meromyosin junction is favored by phosphorylation. These observations may signify a structural effect of phosphorylation on the prevailing interactions within the myosin filament in physiological solvent conditions.