Antipeptide antibodies as probes of subunit-dependent structural changes in the regulatory domain of the gamma-subunit of phosphorylase kinase

Abstract

The γ-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 γ-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 γ-subunit: the isolated γ-subunit, the γ-δ complex and the holoenzyme complex (αβδγ)₄. Of the four antibodies, binding of affinity-purified anti-PhK13 was most affected by alterations in γ-subunit interactions. Taken together, the data from this investigation indicate that the regulatory domain of the γ-subunit can assume different immunochemically distinguishable conformations as the result of interactions among the α-, β-, γ- and δ-subunits of phosphorylase kinase.