Role of magnesium binding to myosin in controlling the state of cross-bridges in skeletal rabbit muscle.
The effect of Mg2+ on the disposition of myosin cross-bridges was studied on myofibrils and synthetic myosin and rod filaments by employing chymotryptic digestion and chemical cross-linking methods. In the presence of low Mg2+ concentrations (0.1 mM), the proteolytic susceptibility at the heavy meromyosin/light meromyosin (HMM/LMM) junction in these three systems sharply increases over the pH range from 7.0 to 8.2. Such a change has been previously associated with the release of myosin cross-bridges from the filament surface [Ueno, H., & Harrington, W.F. (1981) J. Mol. Biol. 149, 619-640]. Millimolar concentrations of Mg2+ block or reverse this charge-dependent transition. Rod filaments show the same behavior as myosin filaments, indicating that the low-affinity binding sites for Mg2+ are located on the rod portion of myosin. The interpretation of these results in terms of Mg2+-mediated binding of cross-bridges to the filament backbone is supported by cross-linking experiments. The normalized rate of S-2 cross-linking in rod filaments at pH 8.0, kS-2/kLMM, increases upon addition of Mg2+ from 0.30 to 0.65 and approaches the cross-linking rate measured at pH 7.0 (0.75), when the cross-bridges are close to the filament surface. In rod filaments prepared from oxidized rod particles, chymotryptic digestion proceeds both at the S-2/LMM junction and at a new cleavage site located in the N-terminal portion of the molecule. Kinetic analysis of digestion rates at these two sites reveals that binding of Mg2+ to oxidized myosin rods has a similar effect at both sites over the pH range from 7.0 to 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002918:Chymotrypsin; D003599:Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D009132:Muscles / Q000378:metabolism*; D009210:Myofibrils / Q000187:drug effects / Q000648:ultrastructure*; D009218:Myosins / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D011817:Rabbits
D010446:Peptide Fragments; D002918:Chymotrypsin; D009218:Myosins; D008274:Magnesium