Regulation of scallop myosin by calcium. Cooperativity and the "off" state.

Abstract

Scallop subfragment 1 (S1) is an unregulated molecule; it differs from heavy meromyosin (HMM) and myosin in that it has no "off" state, although it contains the full complement of light chains and the triggering calcium binding site. S1 differs from myosin by lacking the head-rod junction and being single-headed. The contribution of the head-rod junction was evaluated by studying single-headed myosin. Isolated single-headed myosins show some regulation; their actin activated ATPase is stimulated about 3-fold by calcium. However, in contrast to HMM and myosin, the calcium dependence of ATPase activation of single-headed myosin is non-cooperative. The single ATP turnover rate of single-headed myosin in the absence of calcium is less than 30 seconds (our experimental resolution) compared to the approximately 5 minute turnover rate of myosin. HMM and myosin exhibit several cooperative features not shown by S1. Calcium binding becomes cooperative in the presence of nucleotide analogues in HMM and myosin, but not in S1. Nucleotide analogues are bound cooperatively by myosin and HMM in the absence of calcium; the introduction of calcium to the system reduces the affinity and abolishes the cooperative binding of nucleotide in the double headed molecules. Conversely, S1 shows normal binding curves for nucleotide analogues both in the presence and absence of calcium. Therefore, there is direct communication between the calcium binding sites and nucleotide binding sites in regulated molecules that is mediated by interaction between the two heads. .