Antipeptide antibodies as probes of subunit-dependent structural changes in the regulatory domain of the gamma-subunit of phosphorylase kinase.
The gamma-subunit of phosphorylase kinase contains a protein kinase catalytic domain (residues 20-276) and a regulatory domain (residues 276-386). The purpose of the present investigation was to develop monospecific antibodies against four synthetic gamma-subunit regulatory domain peptides (PhK1: 362-386; PhK5: 342-366; PhK9: 322-346; PhK13: 302-326) to use as probes to study the structure of the regulatory domain. Each affinity-purified antibody was characterized with regard to its ability to bind three different structural forms of the gamma-subunit: the isolated gamma-subunit, the gamma-delta complex, and the holoenzyme complex (alpha beta delta gamma)4. Of the four antibodies, binding of affinity-purified anti-PhK13 was most affected by alterations in gamma-subunit interactions. Taken together, the data from this investigation indicate that the regulatory domain of the gamma-subunit can assume different immunochemically distinguishable conformations as the result of interactions among the alpha-, beta-, gamma-, and delta-subunits of phosphorylase kinase.
Wangsgard
W P
WP
Department of Pharmacology & Toxicology, University of Utah, Salt Lake City 84112, USA.
Dasgupta
M
M
Blumenthal
D K
DK
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D000915:Antibody Affinity; D000918:Antibody Specificity; D000936:Antigen-Antibody Complex; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D010764:Phosphorylase Kinase / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D011817:Rabbits
D000906:Antibodies; D000936:Antigen-Antibody Complex; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010764:Phosphorylase Kinase