Crosslinking of a 28-residue N-terminal peptide of actin to myosin subfragment 1.
The N-terminal 28-residue peptide of actin whose Cys10 was labeled with 5-iodoacetamido-fluorescein (F3K peptide) was isolated from the fluorescently labeled thrombin digest of actin. The effect of myosin subfragment 1 (S1) on the fluorescence of F3K peptide was examined in the absence of ATP. With increasing concentration of S1 added, the fluorescence intensity of F3K peptide increased by maximally 7.3% with an apparent dissociation constant of 5.7 microM, suggesting a role of this peptide region of actin in acto-S1 binding in rigor. F3K peptide was crosslinked with S1 at 10 mM NaCl using a zero-length crosslinker by the method of Grabarek and Gergely [Anal. Biochem. 185, 131-135 (1990)]. The crosslinking was greatly inhibited by the presence of either 0.2 M NaCl or 5 mM MgATP. The analyses of amino acid compositions and sequences of the fluorescent peptides isolated from a lysylendopeptidase digest of the crosslinked S1 indicated that F3K peptide was mainly crosslinked to residues 637-642 of the S1 heavy chain. The crosslinked S1 was isolated by selectively pelleting the uncrosslinked S1 with F-actin. ATPase activity of the isolated crosslinked S1 alone was twice as high as that of control S1. The actin-activated ATPase activity of the crosslinked S1 was much lower than that of uncrosslinked S1. The estimated Vm and Km values were 1.72 s-1 and 125 microM, respectively. The Vm decreased to less than 1/8, while Km increased only twofold. The results suggest that the N-terminal 28-residue segment of actin may be implicated in the rigor binding of actomyosin and in the actin-activation of myosin ATPase, but may not be the main determinant of actomyosin binding in the presence of ATP.
Kunori
S
S
Department of Chemistry, Hokkaido University, Sapporo.
Katoh
T
T
Mogi
Y
Y
Morita
F
F
D000199:Actins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D003545:Cysteine; D010450:Endopeptidases; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D015879:Myosin Subfragments / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000199:Actins; D003432:Cross-Linking Reagents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D010446:Peptide Fragments; C015296:5-iodoacetamidofluorescein; D010450:Endopeptidases; D003545:Cysteine