The complete amino-acid sequence of actin of rabbit skeletal muscle was determined. The actin polypeptide chain is composed of 374 residues, including one residue of the unusual amino acid NT-methyl histidine, and has a calculated molecular weight of 41,785. The sequence of actin was determined by isolating the peptides produced by cleavage of the protein with cyanogen bromide, determining the sequence of these The location and function of actin is most clearly understood in vertebrate skeletal muscle; the actin units are assembled into double-stranded helices which, together with tropomyosin and the troponin complex, constitute the thin filaments. Extensions of myosin molecules in the thick filaments can interact with actin, forming crossbridges between the filaments. During the lifetimes of attachment of these crossbridges, force is generated, which results in sliding of the filaments past each other. Thus, actin serves as the site of crossbridge attachment and also as a support for the regulatory proteins, tropomyosin and troponin.Besides being found in skeletal, smooth, and cardiac muscle, actin-like proteins with properties that are very similar to those of muscle actin have been isolated from several other sources, including ameba (1), blood platelets (2), fibroblasts (3), brain (4), and cytoplasm of numerous embryonic cells (5). The exact role of actin in each of these cell types is under investigation, but the ability of cytoplasmic actin to interact with myosin from muscle (1, 2, 4) implies that in nonmuscle cells it is also involved in force generation.Actin clearly occupies a central role in biological movement, and, as a necessary step toward understanding its function on a molecular level, we have determined the amino-acid sequence of actin isolated from rabbit skeletal muscle. Actin Preparation. Rabbit back and leg muscle was used as a source of actin (6, 7). Purified actin gave a single band on Na dodecyl sulfate-gel electrophoresis. Reduction and alkylation or aminoethylation of sulfhydryl groups have been described (6, 7).Cyanogen Bromide Cleavage. Protein or peptides were dissolved in 70% formic acid and treated with a 50-to 125-fold molar excess of cyanogen bromide (CNBr) over methionine residues for 16-24 hr at 230. Under these conditions, the methionine was usually quantitatively converted to homoserine (6). In some cases complete conversion was insured by treatment of the protein with 2-mercaptoethanol, in order to convert residual methionine sulfoxide to methionine, and again treating the protein with cyanogen bromide (7). Separation of CNBr Peptides. Peptides CB-1 through CB-13 (see Table 1) were first fractionated on Sephadex G-50 equilibrated with 25% acetic (Fig. la) or 20% formic acid and were then purified by ion-exchange chromatography (6-8). The sequences of the purified cyanogen bromide fragments were determined by studying the products of enzymic digestion of the peptides with trypsin, chymotrypsin, and/or thermolysin.Peptides CB-15, CB-16, and CB-17 were separated from...
