title: Purification and characterization of catalytic fragments of phosphorylase kinase gamma subunit missing a calmodulin-binding domain.
abstract: A catalytic fragment preparation of rabbit muscle phosphorylase kinase produced by limited chymotryptic digestion was isolated and identified as the NH2-terminal region of the gamma subunit by Edman degradation. Mass spectral analysis, gas phase sequence analysis, and amino acid analysis of the active fragment carboxyl-terminal peptides revealed multiple COOH termini generated at residues Tyr290, Arg296, and Phe298 in the gamma subunit sequence. These active fragment species are about 24% smaller than the gamma subunit (Mr 44,673) and range in size from Mr 33,279 to Mr 34,275. The active fragment preparation exhibits a specific activity about 6-fold higher than that of the gamma subunit-calmodulin complex. Calmodulin confers calcium sensitivity to the gamma subunit but has no effect on the enzymatic properties of active fragment. Affinity measurements demonstrated a dissociation constant of 0.7 microM for active fragment binding to dansylcalmodulin, a value about 28-fold weaker than reported for the gamma subunit. These data support the presence of a calmodulin binding domain in the COOH-terminal region of the gamma subunit.
authors.affiliation: Protein Chemistry Facility, W. Alton Jones Cell Science Center, Lake Placid, New York 12946.
authors.forename: W R
authors.initials: WR
authors.lastname: Harris
authors.forename: D A
authors.initials: DA
authors.lastname: Malencik
authors.forename: C M
authors.initials: CM
authors.lastname: Johnson
authors.forename: S A
authors.initials: SA
authors.lastname: Carr
authors.forename: G D
authors.initials: GD
authors.lastname: Roberts
authors.forename: C A
authors.initials: CA
authors.lastname: Byles
authors.forename: S R
authors.initials: SR
authors.lastname: Anderson
authors.forename: L M
authors.initials: LM
authors.lastname: Heilmeyer
authors.forename: E H
authors.initials: EH
authors.lastname: Fischer
authors.forename: J W
authors.initials: JW
authors.lastname: Crabb
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D007700:Kinetics; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D010446:Peptide Fragments / Q000302:isolation & purification; D010764:Phosphorylase Kinase / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits
chemical_list: D002147:Calmodulin; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010764:Phosphorylase Kinase; D003488:Cyanogen Bromide