Cleavage at site A, Glu-642 to Ser-643, of the gizzard myosin heavy chain decreases affinity for actin

J Biol Chem. 1993 Dec 5;268(34):25948-51.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actins / metabolism*
  • Amino Acid Sequence
  • Animals
  • Electrophoresis, Polyacrylamide Gel
  • Gizzard, Avian / metabolism
  • Glutamates*
  • Glutamic Acid
  • Kinetics
  • Metalloendopeptidases / metabolism
  • Molecular Sequence Data
  • Muscle, Smooth / metabolism
  • Myosin Subfragments / metabolism*
  • Myosins / chemistry
  • Myosins / metabolism*
  • Papain / metabolism
  • Peptide Fragments / isolation & purification
  • Serine*
  • Substrate Specificity
  • Turkeys

Substances

  • Actins
  • Glutamates
  • Myosin Subfragments
  • Peptide Fragments
  • Glutamic Acid
  • Serine
  • Papain
  • Metalloendopeptidases
  • auR protein, Staphylococcus aureus
  • Myosins