Mapping myosin-binding sites on actin probed by peptides that inhibit actomyosin interaction.
A 2-kDa peptide (2K peptide) which was derived from the neck region of porcine aorta smooth muscle myosin heavy chain binds to actin competitively with skeletal myosin subfragment 1 (S1) in the absence of ATP and inhibits acto-S1 ATPase activity [Katoh, T. and Morita, F. (1993) J. Biol. Chem. 268, 2380-2388]. Using this and other peptides, myosin-binding sites on actin were mapped and their functions were studied. The 2K peptide inhibited the acto-S1 ATPase activity without inhibiting the binding of S1 to actin in the presence of ATP. On the other hand, the dansylated 2K peptide (DNS-2K peptide) inhibited not only the acto-S1 ATPase activity but also the binding of S1 to actin in the presence of ATP. Then, DNS-2K peptide was crosslinked to actin with 1-ethyl-3[3-(dimethylamino)propyl] carbodiimide. Amino acid composition and sequencing analyses of the fluorescent lysylendopeptidase-peptides of the crosslinked product indicated that DNS-2K peptide was crosslinked to acidic residues within residues 1-18 (Asp1, Glu2, Asp3, Glu4, and/or Asp11), 19-50 (Asp25), and 85-113 (Glu99 or Glu100) of actin. A competition experiment for the crosslinking with unlabeled 2K peptide showed that the crosslinking to residues 85-113 of actin was specific for DNS-2K peptide. In addition, isolated actin peptide 85-113 was found to show the competitive inhibition of actin-activated ATPase activity of S1 with respect to actin. These results suggest that the site within residues 1-28 of actin participates in the actin-activation of myosin ATPase activity, and the site within residues 85-113 of actin participates in the weak binding of myosin to actin in the presence of ATP.
Katoh
T
T
Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo.
Morita
F
F
D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000205:Actomyosin / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D003432:Cross-Linking Reagents; D003619:Dansyl Compounds; D007700:Kinetics; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D009218:Myosins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D011817:Rabbits
D000199:Actins; D003432:Cross-Linking Reagents; D003619:Dansyl Compounds; D010446:Peptide Fragments; D000205:Actomyosin; D009218:Myosins