Immunochemical characterization of the gamma-subunit of phosphorylase kinase

Phosphorylase kinase is a multisubunit protein kinase with the subunit composition, (alpha-beta-delta-gamma)4, that is a key regulatory enzyme in glycogenolysis. Catalytic activity is controlled by phosphorylation of its alpha- and beta-subunits and by Ca++ binding to its delta-subunit. The gamma-subunit is the catalytic subunit and interacts directly with these subunits, although the specific molecular interactions taking place are still poorly understood. This research was designed to identify how the subunits of phosphorylase kinase interact and how the catalytic activity of the gamma-subunit is regulated by these interactions using monospecific polyclonal antibodies. Four anti-peptide antibodies--PhK1, PhK5, PhK9, and PhK13--that correspond to the regulatory domain of the gamma-subunit were purified by affinity chromatography. The antibodies were tested for Ca++-dependent binding to different forms of the gamma-subunit using an ELISA. The effect of the antibodies on holoenzyme and gamma-delta complex catalytic activity was also examined. The antibody to PhK1 bound tightly to all forms of the gamma-subunit in a Ca++-independent manner. The presence of anti-PhK1 antibody resulted in a Ca++-independent increase in enzyme activity. The antibody to PhK5 bound weakly to the holoenzyme with a slight enhancement in the presence of Ca++, and bound slightly tighter to the gamma-delta complex and gamma-subunit forms. A Ca++-independent stimulation in enzyme activity was also detected. The anti-PhK9 antibody showed very weak binding to all forms of the gamma-subunit. There was slight inhibition of phosphorylase kinase activity and complete inhibition of gamma-delta complex activity with overnight incubation. Modest binding to the holoenzyme was seen with the anti-PhK13 antibody, which was increased in the presence of Ca++. Slight binding with no Ca++-dependence was seen with the gamma-delta complex and no binding was seen with the gamma-subunit. In the presence of anti-PhK13 complete inhibition of gamma-delta complex activity was seen, and partial inhibition of holoenzyme activity was obtained. The ability of these antibodies to bind to the regulatory domain of the gamma-subunit and alter its catalytic activity has enabled us to better understand the structures and conformational changes that are important for the regulation of phosphorylase kinase activity.