Actin-binding peptides obtained from the C-terminal 24-kDa fragment of porcine aorta smooth muscle myosin subfragment-1 heavy chain.
The C-terminal 24-kDa fragment of myosin subfragment-1 (S-1) heavy chain was isolated by papain digestion of porcine aorta myosin followed by ethanol fractionation. The isolated 24-kDa fragment was digested by lysylendopeptidase. When the digest was ultracentrifuged with F-actin, 7- and 2-kDa peptides coprecipitated with the actin. These two peptides were isolated by high performance liquid chromatography, and their amino acid sequences were determined. The 7- and 2-kDa peptides correspond to residues 692-744 and 835-846, respectively, of the chicken gizzard myosin heavy chain (Yanagisawa, M., Hamada, Y., Katsuragawa, Y., Imamura, M., Mikawa, T., and Masaki, T. (1987) J. Mol. Biol. 198, 143-157). The 7-kDa peptide contains the reactive cysteine residues, SH1 and SH2. The isolated 7- and 2-kDa peptides also bound to F-actin with dissociation constants of 0.6 and 12 microM, respectively. The 2-kDa peptide was found to compete with rabbit skeletal S-1 for the binding to F-actin by examining the binding of S-1 to actin in the presence of various concentrations of the 2-kDa peptide. The 2-kDa peptide was also shown to interact with the regulatory light chain of aorta myosin by difference UV absorption spectroscopy. The 2-kDa peptide inhibited the actin-activated ATPase activity of skeletal S-1, and the inhibition was canceled by the addition of the isolated regulatory light chain. These results suggest that the newly found 2-kDa peptide region may be related to the regulation of smooth muscle actomyosin ATPase activity by phosphorylation of the regulatory light chain.
Katoh
T
T
Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo, Japan.
Morita
F
F
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000737:chemistry; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013056:Spectrophotometry, Ultraviolet; D013552:Swine
D000199:Actins; D008840:Microfilament Proteins; D015879:Myosin Subfragments; D010446:Peptide Fragments