Structural aspects of myosin subfragment 1 as studied by subtilisin digestion of trypsin-split S-1 (27 kDa-50 kDa-20 kDa).
Limited subtilisin digestion of myosin subfragment 1 (S-1) was carried out, varying the enzyme: substrate weight ratio from 1:200 to 1:10, and changes in structure, and in the MgATPase activities of S-1 and acto-S-1 after proteolysis, were followed. When the starting material--tryptically-cleaved S-1 (27 kDa-50 kDa-20 kDa) ("split S-1")--was subjected to further subtilisin digestion, it was found that with increasing enzyme concentration, the 50 kDa fragment degraded into an 18 kDa fragment via a 33 kDa peptide (50----33----18 kDa), which was not cross-linked with F-actin. On the other hand, the 27 and 20 kDa fragments were rather stable at lower subtilisin concentrations and started to degrade only at higher subtilisin concentrations. These degradations lowered the MgATPase activities of S-1 and acto-S-1. The losses of MgATPase activities of S-1 and of acto-S-1 were mainly due to the degradations of the 27 and 20 kDa fragments, respectively. Addition of EDTA did not affect the subtilisin cleavage pattern of split S-1 but the breakdown of the 50 kDa fragment was extremely depressed, suggesting that some conformational change of the 50 kDa fragment is induced by the binding of divalent cation. The binding of MgADP to split S-1 accelerated the degradation of the 27 kDa fragment and produced a new cut in the 27 kDa fragment (27----20 kDa), resulting in a further loss of the S-1 MgATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D015879:Myosin Subfragments; D009218:Myosins / Q000302:isolation & purification* / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D013381:Subtilisins; D014357:Trypsin
D015879:Myosin Subfragments; D010446:Peptide Fragments; D013381:Subtilisins; D014357:Trypsin; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins