Purification from bovine liver membranes of a guanine nucleotide-dependent activator of phosphoinositide-specific phospholipase C. Immunologic identification as a novel G-protein alpha subunit.

Abstract

Cholate-solubilized extracts from bovine liver plasma membranes preincubated with the nonhydrolyzable GTP analog guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) displayed enhanced phosphoinositide-specific phospholipase C activity compared with extracts from membranes incubated without nucleotide or with ATP or GDP analog. Resolution of the GTP gamma S-elicited activator of phospholipase C was achieved using heparin-Sepharose which bound the phospholipase C activity. Recombination of non-adsorbed extract with salt-eluted phospholipase C activity resulted in a stimulation of enzyme activity. The GTP gamma S-dependent activator was purified, on the basis of its ability to activate partially purified phospholipase C, by sequential chromatography on Q-Sepharose, Sephacryl S-300, octyl-Sepharose, and Mono Q. The presence of G-protein beta subunits and the alpha subunits of Gi1, Gi2, and Gi3 was detected, by immunoblot analysis, in Mono Q-purified phospholipase C activator preparations. Resolution of the activator from these alpha subunits was achieved by incubation with pertussis toxin in the presence of millimolar NAD+ followed by rechromatography on Mono Q. The phospholipase C activator, thus resolved from ADP-ribosylated alpha i subunits, possessed an approximate Mr of 42 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and copurified with a substoichiometric amount of beta subunit. Immunoblot analysis of fractions from the final Mono Q column revealed cross-reactivity of the 42-kDa phospholipase C activator with antipeptide antibodies raised against residues 160-169 of alpha i1 and a region of sequence common to all known G-protein alpha subunits. The 42-kDa activator was not recognized by other alpha subunit-specific or common antibodies. These findings identify the purified phospholipase C activator as a novel G-protein alpha subunit. This may represent the active subunit of the pertussis toxin-insensitive G-protein mediating receptor-stimulated phosphoinositide breakdown in mammalian liver.