Cloning and characterization of the S1 domain of four myosin isoforms fromfunctionally divergent fiber types in adult Rana pipiens skeletal muscle

The motor properties of myosin reside in the globular S1 region of the myos in heavy chain (MHC) subunit. All vertebrates express a family of MHC isofo rms in skeletal muscle that have a major influence on the mechanical proper ties of the various fiber types. Differences in molecular composition of S1 among MHC isoforms within a species have not been studied to any great det ail. Presently, we have isolated, cloned and sequenced the S1 subunit of fo ur MHC isoforms from skeletal muscle in Rana pipiens that are specifically expressed in four mechanically divergent fiber types. Paired analysis showe d that the overall amino acid identity was higher between the three S1 isof orms expressed in twitch fibers than between the twitch and tonic isoforms. Relatedness in amino acid composition was evaluated in regions reported to govern cross-bridge kinetics. Surface loops 1 and 2, thought to influence motor velocity and ATPase, respectively, were both highly divergent between isoforms. However, the divergence in the loops was roughly equal to that o f the amino-terminal region, a domain considered less important for motor f unction. We tested the hypothesis that the loops are more conserved in pair s of isoforms with more similar kinetics. Comparisons including other verte brate species showed no tendency for loops from pairs with similar kinetics to be more conserved. These data suggest that the overall structure of loo ps 1 and 2 is not critical in regulating the kinetic properties of R. pipie ns S1 isoforms. Cloning of this family of frog S1 isoforms will facilitate future structure/function studies of the molecular basis of variability in myosin cross-bridge kinetics. (C) 2000 Published by Elsevier Science B.V. A ll rights reserved.