Regulatory light-chains and scallop myosin: Full dissociation, reversibility and co-operative effects

Abstract

Both regulatory light-chains of scallop myosin are removed from myofibrils by a short EDTA treatment at elevated temperatures (25°C in the case of Placopecten magellanicus; 35°C in the case of Aequipecten irradians). Essential light-chains are not lost by this treatment. Denuded myofibrils readily recombine with regulatory light-chains in the presence of magnesium ions up to a stoichiometry of two moles of regulatory light-chains per mole of myosin. High-affinity, specific calcium binding sites are proportional to regulatory light-chain content and are lost upon complete removal of regulatory light-chains. Denuded myofibrils contain two non-specific divalent cation binding sites of lower affinity per myosin that are not found on intact myofibrils. Occupancy of these sites by calcium depresses the actin-activated Mg2+-ATPase activitybby about 20 to 30%. Regulatory light-chains specifically modify the actin-activated Mg2+-ATPase of scallop myofibrils, inhibit the ATPase 10 to 20-fold in the absence of calcium but increase the turnover rate by two to fourfold if calcium is present. The elevated Mg2+-ATPase of denuded myofibrils in the absence of calcium is restored biphasically by regulatory light-chains to the low rates typical of intact myofibrils. This ATPase rate remains constant until one mole of regulatory light-chain is added back, then is depressed linearly upon further re-addition of regulatory light-chains. This ATPase dependency is taken as evidence that both regulatory light-chains must be attached to intact scallop myosin for regulation, and suggests that regulatory light-chains re-add in a negatively co-operative manner.