Comparative Study of Dogfish and Rabbit Muscle Phosphorylases

S-Carboxymethylated, NaBH₄-reduced, 32P-labeled dogfish phosphorylase a was cleaved with cyanogen bromide and the 32P and phosphopyridoxyl-containing fractions were separated by a combination of gel filtrations and ion-exchange chromatographies. The 32P-labeled CNBr peptide was very similar to the corresponding peptide from rabbit phosphorylase and on digestion with chymotrypsin, gave a radioactive phosphopeptide, Glu-Arg-Arg-Lys-Gln-Ile-Ser(P)-Val-Arg-Gly-Leu, with a sequence identical with the phosphopeptide isolated from rabbit muscle phosphorylase except for the conservative substitution of an arginine for a lysine at position 2. Nine small CNBr peptides were isolated from the phosphopyridoxyl fraction. Two of these together yielded a unique sequence comprising the pyridoxal-5′-P binding site, Met-Lys(Pxy)-Phe-Met-Gly-Arg-Thr-Leu-Gln-Asn-Thr-Met, which is probably identical with the corresponding segment in rabbit muscle phosphorylase. Of the remaining 7 peptides, 5 (44 residues) were identical in composition and partial sequence with peptides derived from rabbit muscle phosphorylase; a sixth peptide was probably homologous with another rabbit octapeptide. The seventh peptide, which was absent from rabbit phosphorylase, had the sequence, N-acetyl-Ser-Lys-Pro-Lys-Ser-Asp-Met, and was confirmed as the NH₂-terminal sequence of the protein by isolation of the tetrapeptide N-acyl-Ser-Lys-Pro-Lys from a tryptic peptide map of native dogfish phosphorylase. Based on the partial or total sequence of eight homologous CNBr peptides, the distribution of methionine residues, the phosphopeptide sequence, the composition of the CNBr phosphopeptides and that of the whole protein (865 residues), the overall homology between dogfish and rabbit muscle phosphorylases appears to be 85–90%. From the known time of divergence of the dogfish from the main vertebrate line leading to mammals (ca. 450 million years), this corresponds to 1.1–1.7 amino acid mutations/100 residues per 100 million years, a slower rate than found thus far for other proteins with the exception of histones. The results provide a structural basis for the remarkable similarities in physical and regulatory properties between these two proteins described previously (Cohen, P., Duewer, T., and Fischer, E. H. (1971), Biochemistry 10, 2683).