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 ca talytic domain (residues 20-276) and a regulatory domain (residues 276 -386). The purpose of the present investigation was to develop monospe cific antibodies against four synthetic gamma-subunit regulatory domai n peptides (PhK1: 362-386; PhK5: 342-366; PhK9: 322-346; PhK13: 302-32 6) to use as probes to study the structure of the regulatory domain. E ach affinity-purified antibody was characterized with regard to its ab ility to bind three different structural forms of the gamma-subunit: t he 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 ga mma-subunit interactions. Taken together, the data from this investiga tion indicate that the regulatory domain of the gamma-subunit can assu me different immunochemically distinguishable conformations as the res ult of interactions among the alpha-, beta-, gamma-, and delta-subunit s of phosphorylase kinase. (C) 1997 Academic Press