Purification and characterization of catalytic fragments of phosphorylase kinase gamma subunit missing a calmodulin-binding domain.
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.
Harris
W R
WR
Protein Chemistry Facility, W. Alton Jones Cell Science Center, Lake Placid, New York 12946.
Malencik
D A
DA
Johnson
C M
CM
Carr
S A
SA
Roberts
G D
GD
Byles
C A
CA
Anderson
S R
SR
Heilmeyer
L M
LM
Fischer
E H
EH
Crabb
J W
JW
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
D002147:Calmodulin; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010764:Phosphorylase Kinase; D003488:Cyanogen Bromide