Purification of an A1F₄-^- and G-protein βγ- subunit-regulated phospholipase C-activating protein

A 150-kDa phospholipase C has previously been purified from turkey erythrocytes and has been shown by reconstitution with turkey erythrocyte membranes to be a receptor- and G-protein-regulated enzyme (Morris, A. J., Waldo, G. L., Downes, C. P., and Harden, T. K. (1990) J. Biol. Chem. 265, 13501-13507; Morris, A. J., Waldo, G. L., Downes, C. P., and Harden, T. K. (1990) J. Biol. Chem. 265, 13508-13514). Combination of this 150-kDa protein with phosphoinositide substrate-containing phospholipid vesicles prepared with a cholate extract from purified turkey erythrocyte plasma membranes resulted in conferrence of AlF₄^- sensitivity to the purified phospholipase C. Guanosine 5′-3-O-(thio)triphosphate also activated the reconstituted phospholipase C in a manner that was inhibited by guanosine 5′-2-O-(thio)-diphosphate. The magnitude of the AlF₄^- stimulation was increased with increasing amounts of plasma membrane extract, and was also dependent on the concentration of purified phospholipase C. Using reconstitution of AlF₄^- sensitivity as an assay, the putative G-protein conferring regulation to the 150-kDa phospholipase C was purified to near homogeneity by sequential chromatography over Q-Sepharose, Sephacryl S-300, octyl-Sepharose, hydroxylapatite, and Mono-Q. Reconstituting activity co-purified with an approximately 43-kDa protein identified by silver staining; lesser amounts of a 35-kDa protein was present in the final purified fractions, as was a minor 40-kDa protein. The 43-kDa protein strongly reacted with antiserum against a 12-amino acid sequence found at the carboxyl terminus of G_q and G₁₁, the 35-kDa protein strongly reacted with G-protein β-subunit antiserum, and the 40-kDa protein reacted with antiserum that recognizes G_i3. Immunoprecipitation of the 43-kDa protein resulted in loss of phospholipase C-stimulating activity of the purified fraction. The idea that this is a phospholipase C-regulating G-protein is further supported by the observation that co-reconstitution of G-protein βγ-subunit with the purified phospholipase C-activating fraction resulted in a βγ-subunit-dependent inhibition of ALF₄^--stimulated phospholipase C activity in the reconstituted preparation.