Structural Differences in the Crossbridge Head of Temperature‐Associated Myosin Subfragment‐1 Isoforms from Carp Fast Skeletal Muscle

We determined the primary structures of the three acclimation‐temperature‐associated isoforms of myosin subfragment‐1 heavy chain from fast skeletal muscle of thermally acclimated carp. These isoforms were cloned by extending 5′‐regions of cDNAs that encode the rod part of myosin heavy chain specifically expressed in 10°C‐ and 30°C‐acclimated carp, together with the region that encodes an intermediate structure [Imai, J., Hirayama, Y., Kikuchi, K., Kakinuma, M. & Watabe, S. (1997) J. Exp. Biol. 200 , 27–34]. These three isoforms generally resembled each other in primary structure, showing 94.8, 90.9, and 92% similarity between the 10°C‐ and intermediate‐type, between the 10°C‐ and 30°C‐type, and between the intermediate‐ and 30°C‐type myosin heavy chains, respectively. However, isoform‐specific differences were clearly observed between the 10°C‐ and 30°C‐type heavy chains in the first 60 amino acid residues from the N‐terminus, where the intermediate‐type showed an intermediate feature in its sequence compared to the 10°C‐ and 30°C‐type isoforms. Other striking differences were observed in two surface loops between the 10°C‐ and 30°C‐type isoform. Five amino acid residues out of sixteen were different in loop 1 near the ATP‐binding pocket, and six out of twenty were different in loop 2 on the actin‐binding site. The loops connecting β‐sheets that are known to surround the ATP‐binding pocket were highly conserved in primary structure for the three types. In northern blot analysis, the accumulated mRNA levels of the 10°C‐ and intermediate‐type isoforms were significantly higher in carp acclimated to 10°C and 20 °C than carp acclimated to 30 °C, whereas the level of the 30°C‐type isoform was significantly higher in carp acclimated to 30°C than those acclimated to 10°C and 20°C.