CROSS-LINKING 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 mu M, 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 SI was much lower than that of uncrosslinked S1. The estimated V-m and K-m values were 1.72 s(-1) and 125 mu M, respectively, The V-m decreased to less than 1/8, while K-m 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.