Phosphorylation and the binding of calcium and magnesium to skeletal myosin.
It has previously been shown that the binding of calcium and magnesium ions to the isolated metal-binding light 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 calcium ions bind to the light chain in situ on the myosin head. Moreover the affinity for calcium 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 cooperativity. The affinity of the site for both calcium and magnesium ions is enhanced by 1-2 orders of magnitude when the light chain in incorporated in the myosin heads. The effect of phosphorylation on the affinity persists in these circumstances, being marked for calcium and small for magnesium. On phosphorylation the calcium binding constant falls from 8 x 10(6) M-1 to 4 x 10(6) M-1 at physiological ionic strength, compared with 2.5 x 10(5) M-1 and 5 x 10(4) M-1 for the isolated light chains. The sensitivity of the proteolytic cleavage sites is affected by phosphorylation. Thus in the absence of calcium ions the yield of subfragment 1 at a low chymotrypsin concentration is substantially greater in dephosphorylated than phosphorylated myosin, whereas at saturating concentrations of calcium ions attack at the light meromyosin/heavy meromyosin junction is favoured by phosphorylation. These observations may signify a structural effect of phosphorylation on the prevailing interactions within the myosin filament in physiological solvent conditions.
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002918:Chymotrypsin; D002942:Circular Dichroism; D008274:Magnesium / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D011817:Rabbits
D002918:Chymotrypsin; D009218:Myosins; D008274:Magnesium; D002118:Calcium