Both the 25-kDa and 50-kDa domains in myosin subfragment 1 are close to the reactive thiols (benzophenone-4- Communicated by Russell F. Doolittle, May 23, 1986 ABSTRACT The thiol-specific photoactivatable reagent benzophenone-4-iodoacetamide can be incorporated into myosin subfragment 1 (Si), accompanied by an increase of Ca2+-ATPase and the loss of K+-ATPase activities, a characteristic property of S1 when reactive sulfhydryl 1 (SH-1) Is modified. After trypsin cleavage, 25-kDa, 50-kDa, and 20-kDa fragments were found upon NaDodSO4/polyacrylamide gel electrophoresis of the unphotolyzed sample, whereas only the 50-kDa fragment and a 45-kDa fragment appeared In the photolyzed sample, indicating that the NH2-terminal 25-kDa fragment was crosslinked to the COOH-terminal 20-kDa fragment via SH-1. When photolysis 'was carried out in the presence of Mg2+ and ATP or Mg2e and adenosine 5-[,B, imidoltriphosphate (AdoPP[NHJP), a 70-kDa band, attribu-

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The thiol-specific photoactivatable reagent benzophenone iodoacetamide (BPIA) can be selectively incorporated into the most reactive thiol, SH-1, of myosin S1, and upon photolysis, an intramolecular cross-link is formed between SH-1 and the N-terminal 25-kDa region of S1. If a Mg2+-nucleotide is present during photolysis, cross-links can be formed either with the 25-kDa region or with the central 50-kDa region [Lu et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6392]. Comparison of the peptide maps of cross-linked and un-cross-linked S1 heavy chains indicates that the segment located about 12-16 kDa from the N-terminus of the heavy chain can be cross-linked to SH-1 via BPIA independently of the presence of a nucleotide whereas the segment located 57-60 kDa from the N-terminus can be cross-linked to SH-1 only in the presence of a Mg2+-nucleotide [Sutoh & Lu (1987) Biochemistry 26, 4511]. In this report, S1 was labeled with radioactive BPIA, photolyzed in the absence of nucleotide, and then degraded with proteolytic enzymes. Peptides containing cross-links were isolated by liquid chromatography and subjected to amino acid sequence analyses. The results show that Glu-88 is the major site and Asp-89 and Met-92 are the minor sites involved in cross-linking with SH-1 (Cys-707) via BPIA. These residues are very near the reactive lysine residue (Lys-83) but relatively remote in the primary structure from the putative nucleotide binding region.