Sequence variations and turnover rates in molluscan myosin isoforms.

摘要

Molluscan myosins are regulated directly by their light chain subunits. We studied several molluscan myosin isoforms to determine the contribution of the subunits, and specific residues, to their enzymatic activities. Striated muscle myosins of the sea scallop, Placopecten, have higher ATPase activities than those of its catch muscles, or of the striated muscle of Argopecten, a bay scallop. The motility of these isoforms varied in a similar manner. To determine which subunits cause these differences, we cloned and sequenced the cDNA encoding the essential light chains (ELC), regulatory light chains (RLC) and heavy chains (MHC) of Placopecten striated and catch muscle myosins. The ELC of both muscle types of Placopecten were identical, and highly homologous to the Argopecten ELC. Therefore, the ELC does not contribute to the different properties of these myosins. Three RLC isoforms were present in Placopecten, two in catch muscle and one in striated, that are alternatively spliced from a single RLC gene. Hybrid studies indicated that these RLC isoforms do not modulate the enzymatic activities. One RLC isoform encoded a putative consensus sequence for phosphorylation by smooth muscle myosin light chain kinase. Hybrids containing the phosphorylated RLC had the same enzymatic activity as the ones reconstituted with the unphosphorylated RLC. MHC sequences showed two muscle-specific MHC isoforms. They are spliced variants with 98% overall identity, and differences restricted to small regions of the sequence. The most divergent regions were in the rod portion. ATPase activities of subfragment 1 from these myosins showed differences in activity similar to whole myosins, indicating that myosin head sequence variations were responsible for the modulation of enzymatic activity. These isoforms differ in turnover rates, not in actin affinity. The surface loop near the nucleotide binding site is the only area in which all the MHC sequences diverge. In summary, we provide evidence to suggest that the different turnover rates and velocities of movement of several myosin isoforms are due to amino acid sequence variability within the surface loop near the nucleotide binding site.