Effect of mild heat treatment on the ATPase activity and proteolytic sensitivity of myosin subfragment-1.
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 degrees C. Addition of F-actin, ATP, or ATP analogs, such as ADP or PPi, to S-1 before incubation at 35 degrees 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 degrees 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.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D002918:Chymotrypsin / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006358:Hot Temperature; D006868:Hydrolysis; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011489:Protein Denaturation; D014357:Trypsin / Q000378:metabolism
D015879:Myosin Subfragments; D010446:Peptide Fragments; D004492:Edetic Acid; D002918:Chymotrypsin; D014357:Trypsin; D000251:Adenosine Triphosphatases; D009218:Myosins