title: Identification and primary structure of calmodulin binding domains in the phosphorylase kinase holoenzyme.
abstract: Fast twitch skeletal muscle phosphorylase kinase was isolated and incubated with a radioactive, bifunctional, photoactivable, and cleavable cross-linker conjugated to calmodulin. Incubation of the holoenzyme only resulted in the labeling of the alpha-subunit in the presence of Ca2+. After cleavage with CNBr (and subdigestion with Asp-N protease), a sequence was identified (residues 1069-1087) in the alpha-subunit which had the predominant basic character and the propensity to form an amphiphilic helix like other calmodulin binding domains. If cross-linked calmodulin was incubated with the isolated subunits of phosphorylase kinase, radioactivity was recovered in seven CNBr peptides: three came from the alpha-subunits, one of them corresponding to the sequence labeled in the holoenzyme. Three came from the beta-subunit, and one came from the gamma-subunit. The latter contained the two adjacent calmodulin binding domains recently identified in the gamma-subunit (Dasgupta, M., Honeycutt, T., and Blumenthal, D. K. (1988) J. Biol. Chem. 264, 17156-17163).
authors.affiliation: Department of Biochemistry, University of Dundee, Scotland.
authors.forename: P
authors.initials: P
authors.lastname: James
authors.forename: P
authors.initials: P
authors.lastname: Cohen
authors.forename: E
authors.initials: E
authors.lastname: Carafoli
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification; D010764:Phosphorylase Kinase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010764:Phosphorylase Kinase; D003488:Cyanogen Bromide