Isolation and characterization of glycogen phosphorylase b from chicken breast muscle: comparison with a protein extracted from the M. line.

Glycogen phosphorylase b has been purified from chicken breast muscle to a state of homogeneity as judged by the criteria of polyacrylamide gel electrophoresis and ultracentrifugal analysis. The absorbance index A I$ nm was found to be 13.2, the sedimentation coefficient s~(,,~. was 8.7, at a concentration of 2.5 mglml, the subunit molecular weight was 100,000, and the molecular weight of the native enzyme 180,000. In each of these properties, and in its electrophoretie mobility, the chicken muscle enzyme closely resembled its rabbit muscle counterpart. Differences between the chicken and rabbit muscle enzymes were observed in their amino acid compositions and in their specific activities. The chicken muscle enzyme had a specific activity of 72 mg compared to 88 mg for rabbit muscle phopshorylase. The concentration of phosphorylase b in chicken breast muscle was only 15% of that found in rabbit skeletal muscle. The conversion of chicken phosphorylase b to a by rabbit muscle phopshorylase kinase resulted in the incorporation of 1 molecule of phosphate per subunit. Phosphorylase b from chicken muscle showed immunological identity with the I”il, = 100,000 putative M-line protein termed protein A (B. I,. Eaton and F. A. Pepe (1972) J. Cell Biol. 55, 681-695) or component II (T. Masaki and 0. Takaiti (1974) J. Biochem. (Tokyo) 75, 367-380). The proteins were also identical in their content of pyridoxal 5’phosphate (1 molecule per subunit) and in their electrophoretie mobility and stability. It is concluded that the M, = 100,000 M-line protein is phosphorylase. Localization studies, by means of the indirect immunofluorescence method, showed that phosphorylase was tightly bound to isolated washed myofibrils. The fluoresence was mainly located in the I-band region, but was also present in the H-zone, where the M-line is located. The results indicate that phosphorylase is not exclusively an M-line protein, but is also bound to additional myofibrillar proteins in the thin filaments. The significance of the interaction of phosphorylase with the structural proteins of muscle is discussed.