Electrophoretic purification of the alpha and beta subunits of phosphorylase kinase and evidence in support of the deduced amino acid sequences.
A simple, rapid sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method is presented for isolating the alpha, alpha' and beta subunits of rabbit muscle phosphorylase kinase. The SDS-PAGE procedure can yield milligram amounts of alpha and beta from a single preparative gel and also allows isolation of the alpha' isozyme free of alpha. Notably the method provides the purified subunits in a form amenable to structural analysis. Edman degradation of alpha and alpha' reveal identical NH2-terminal structures. Amino acid analysis of the electrophoretically purified alpha and beta subunits are in good agreement with their deduced primary structures. The amino acid sequence of 488 residues in alpha and 713 residues in beta were determined by gas phase Edman degradation. The data support the recently deduced primary structures of alpha (Zander et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 9381-9385).
Crabb
J W
JW
Protein Chemistry Facility, W. Alton Jones Cell Science Center, Lake Placid, NY 12946.
Harris
W R
WR
Johnson
C M
CM
Sotiroudis
T G
TG
Kuhn
C C
CC
Heilmeyer
L M
LM
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification*; D010764:Phosphorylase Kinase / Q000302:isolation & purification*
D010446:Peptide Fragments; D010764:Phosphorylase Kinase