Effect of mild heat treatment on the ATPase activity and proteolytic sensitivity of myosin subfragment-1

Abstract

The K +-EDTA-activated ATPase activity of chymotryptic myosin subfragment-1 (S-1) decreased by 85–90% when S-1 was incubated over a 2-h period at 35 °C. Addition of F-actin, ATP, or ATP analogs, such as ADP or PP i, to S-1 before incubation at 35 °C prevented the loss of ATPase activity. The decrease in ATPase activity was also accompanied by changes in tryptic sensitivity. Instead of the normal peptide pattern—which is comprised of three heavy chain fragments (27K, 50K, and 20K)—only two fragments (27K and 20K) appeared on the sodium dodecyl sulfate-gel electrophoregram after limited tryptic digestion of thermally treated S-1. Addition of any ligand—e.g. ATP, ADP, pyrophosphate, or actin—which prevented the loss of ATPase activity during incubation at 35 °C also prevented the observed change in the tryptic peptide pattern of S-1. Tryptic digested S-1, whose heavy chain has been cleaved to 27K, 50K, and 20K fragments, also lost its ATPase activity upon mild heat treatment. The heat-treated trypsin-digested S-1 was subjected to a second tryptic digestion, which resulted in the disappearance of the 50K fragment, while the 50K fragment of tryptic S-1 not subjected to heat treatment was not susceptible to additional tryptic hydrolysis. The results indicate that the structural changes, that take place specifically in the 50K region of S-1 upon mild heat treatment, lead to both the loss of the ATPase activity and the changed tryptic sensitivity of S-1.