Activation and inhibition of phosphorylase kinase by monospecific antibodies raised against peptides from the regulatory domain of the gamma-subunit.
The C terminus of the catalytic gamma-subunit of phosphorylase kinase comprises a regulatory domain that contains regions important for subunit interactions and autoinhibitory functions. Monospecific antibodies raised against four synthetic peptides from this region, PhK1 (362-386), PhK5 (342-366), PhK9 (322-346), and PhK13 (302-326), were found to have significant effects on the catalytic activities of phosphorylase kinase holoenzyme and the gamma delta complex. Antibodies raised against the very C terminus of the gamma-subunit, anti-PhK1 and anti-PhK5, markedly activated both holoenzyme and the gamma delta complex, in the presence and absence of Ca2+. In the presence of Ca2+ at pH 8.2, anti-PhK1 activated the holoenzyme more than 11-fold and activated the gamma delta complex 2.5-fold. Activation of the holoenzyme and the gamma delta complex by anti-PhK5 was 50-70% of that observed with anti-PhK1. Prior phosphorylation of the holoenzyme by the cAMP-dependent protein kinase blocked activation by both anti-PhK1 and anti-PhK5. Antibodies raised against the peptides from the N terminus of the regulatory domain, anti-PhK9 and anti-PhK13, were inhibitory, with their greatest effects on the gamma delta complex. These data demonstrate that the binding of antibodies to specific regions within the regulatory domain of the gamma-subunit can augment or inhibit structural changes and subunit interactions important in regulating phosphorylase kinase activity.
Wangsgard
W P
WP
Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah 84112, USA.
Meixell
G E
GE
Dasgupta
M
M
Blumenthal
D K
DK
D000595:Amino Acid Sequence; D000906:Antibodies / Q000494:pharmacology*; D001666:Binding Sites, Antibody; D002384:Catalysis; D004789:Enzyme Activation; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology*; D010764:Phosphorylase Kinase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation
D000906:Antibodies; D010455:Peptides; D010764:Phosphorylase Kinase