Abstract
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 Gq and G11, the 35-kDa protein strongly reacted with G-protein beta-subunit antiserum, and the 40-kDa protein reacted with antiserum that recognizes Gi3. 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 beta gamma-subunit with the purified phospholipase C-activating fraction resulted in a beta gamma-subunit-dependent inhibition of AlF(4-)-stimulated phospholipase C activity in the reconstituted preparation.
Highlights
In the absence of AIF; essentially no inositol lipid hydrolysis was observed even with the reconstitution of 300 ng of the purified 150-kDa phospholipase C per assay (Fig. 4)
Tein strongly reacted with G-protein &subunit antiprotein cDNA cloned from a mouse brain library [13].This serum, and the 40-kDa protein reacted with antiserpuromtein, when combined with a partially purified phospholithat recognizes Gi3
We report that thipsurified G-prot.ein-regulated phospholipase C canbe used in an assay to identify and purify a 43,000-dalton protein in Na cholate extracts from turkey erythrocyte plasma membranes that when reconstituted with substrate-containing phospholipidvesicles confers AIFY- and G-protein /3y-subunit sensitivity to the reconstitutedpurified phospholipase C
Summary
In the absence of AIF; essentially no inositol lipid hydrolysis was observed even with the reconstitution of 300 ng of the purified 150-kDa phospholipase C per assay (Fig. 4). Purified turkey erythrocyte plasma membranes were prepared anda Na cholate extract generated as described under “Experimental Procedures.’’ It was found in preliminary experiments that there was a rapid time-dependent loss of capacity of the Na cholate extract to confer AlF; sensitivity to purified 150-kDa phospholipase C, and thatmaintenance of the extract in AMF (see “Experimental Procedures”)greatly reduced this loss of activity.
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