Immunochemical studies on muscle proteins

Abstract

Myosin, tropomyosin A, and tropomyosin B, 3 structure proteins of muscle, were studied. Ouchterlony plates were used for testing the hemogeneity of the preparations, and quantitative precipitin reactions yielded information on the titer of the sera and the stoichiometry of the reaction. Tropomyosin A prepared from clam muscle appears to be a single antigen. Rabbit sera contained up to 492 /xg N of anti-clam tropomyosin A and antibodies localized in the gamma globulin fraction. At equivalence, 2 antibody molecules react with 1 tropomyosin A molecule, while, in the antibody excess region, 1 tropomyosin A molecule is capable of reacting with 4 antibody molecules. Myosin prepared from cat muscle appeared to be homogeneous in the ultracentrifuge. In the agar diffusion experiments, however, multiple lines were observed when cat myosin reacted with anti-cat myosin rabbit sera. Anti-clam tropomyosin A rabbit sera precipitated cat myosin before but not after these sera were allowed to react with clam tropomyosin A. The supernatants of the mixtures of anti-clam tropomyosin A sera and cat myosin showed a reduced ability to react with clam tropomyosin A. From this, the degree of crossover may be estimated at more than 50%. Similarly, anti-cat myosin sera precipitated clam tropomyosin A, but, due to the complexity of the anti-cat myosin antibodies, the degree of crossover could not be estimated. Rabbits could be immunized with tropomyosin A, tropomyosin B, and myosin prepared from rabbit muscle. Significant titers were observed in the sera. Tropomyosin A prepared from rabbit myosin was precipitated by the anti-rabbit myosin sera, and this reaction could be utilized to assess the titer of the antimyosin sera. The results are discussed in terms of the subunit structure of myosin.