Preparation of subfragment-1 from abalone smooth muscle myosin.
Myosin subfragment-1 (S1), which has one heavy chain (HC) (93 kDa) and two light chains (LC1 and LC2), was prepared by papain digestion of myosin from abalone-smooth muscle in the presence of Ca2+. The Ca-sensitivity of abalone S1 itself was not lost completely (about 30%). The tryptic digestion of S1 showed that in the presence of EDTA, S1 HC was split into 68, 55, and 23 kDa fragments, as in the presence of Ca2+, but 23 kDa was further degraded into 19 kDa. In contrast to the result in the presence of Ca2+, LCs disappeared in the early stage of reaction and Ca-ATPase activity decreased rapidly to about 70% of that of intact S1. This rapid decrease of Ca-ATPase activity seemed to be accompanied with the digestion of LCs. Therefore, LCs contribute to the protection of 23 kDa fragment from further digestion, to the maintenance of Ca-ATPase activity by stabilizing the structure of S1 to some extent in the presence of Ca2+. Since F-actin suppressed the cleavage of S1 HC to 68 and 23 kDa during tryptic digestion, it might be that 23 and 68 kDa corresponded to 20 kDa (C-terminal fragment) and to 50 + 25 kDa (N-terminal fragment) of skeletal myosin S1, respectively.
Asakawa
T
T
Hokkaido University of Education, Asahikawa.
Azuma
N
N
D000199:Actins; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D008974:Mollusca / Q000032:analysis*; D009130:Muscle, Smooth / Q000032:analysis*; D015879:Myosin Subfragments; D009218:Myosins / Q000302:isolation & purification*; D010206:Papain; D010446:Peptide Fragments / Q000302:isolation & purification*; D014357:Trypsin
D000199:Actins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D014357:Trypsin; D010206:Papain; D000251:Adenosine Triphosphatases; D009218:Myosins; D002118:Calcium