PMID: 2120213Oct 5, 1990Paper

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.

The Journal of Biological Chemistry
S J TaylorJ H Exton

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 ...Continue Reading

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