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.

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+.