Cleavage at site A, Glu-642 to Ser-643, of the gizzard myosin heavy chain decreases affinity for actin.
The actin-activated ATPase activities of subfragment 1 (S1) produced from gizzard myosin by papain or Staphylococcus aureus protease are different. The activity of the latter is lower, in spite of the presence of intact 20,000-dalton light chains. To study this difference, the S. aureus protease S1 was subjected to further proteolysis by papain. This second stage of proteolysis markedly increased actin-activated ATPase, due to a decrease in K(actin) with no change in Vm and increased the affinity of S1 for actin in the presence of ATP. Treatment with papain caused degradation of the 20-kDa light chain, a decrease in the 26-kDa C-terminal domain of S1 and the 68-kDa fragment containing the N-terminal and central domains, and in the appearance and progressive increase of a 94-kDa fragment. The increase in actin-activated ATPase activity was due to the production of the 94-kDa fragment but not due to light chain degradation. Analyses of N-terminal sequences following papain digestion showed that the 94-kDa fragment was formed from a combination of the 68- and 26-kDa fragments. The bond formed probably involved the N-terminal residue of the 26-kDa fragment (Ser-643) and a side chain carboxyl (Glu-642) or amine (Glu-636). From the sequence data site A was identified as Glu-642-Ser-643. These results confirm the importance of site A in actin-binding of gizzard myosin. It is suggested that the sequence Ser-643 and Val-659, as well as the 3 lysine residues, are important for actin binding.
Ikebe
M
M
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970.
Mitra
S
S
Hartshorne
D J
DJ
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005895:Gizzard, Avian / Q000378:metabolism; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism; D015879:Myosin Subfragments / Q000378:metabolism*; D009218:Myosins / Q000737:chemistry / Q000378:metabolism*; D010206:Papain / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D012694:Serine; D013379:Substrate Specificity; D014422:Turkeys
D000199:Actins; D005971:Glutamates; D015879:Myosin Subfragments; D010446:Peptide Fragments; D018698:Glutamic Acid; D012694:Serine; D010206:Papain; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus; D009218:Myosins