Purification of an AlF4- and G-protein beta gamma-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 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.
Waldo
G L
GL
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill 27599.
Boyer
J L
JL
Morris
A J
AJ
Harden
T K
TK
D000535:Aluminum / Q000302:isolation & purification / Q000378:metabolism*; D017607:Aluminum Compounds; D000818:Animals; D015153:Blotting, Western; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004912:Erythrocytes / Q000201:enzymology; D005459:Fluorides / Q000302:isolation & purification / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D010716:Phosphatidylinositols / Q000378:metabolism; D011233:Precipitin Tests; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D014422:Turkeys; D010738:Type C Phospholipases / Q000378:metabolism*
D017607:Aluminum Compounds; D010716:Phosphatidylinositols; D011506:Proteins; D000535:Aluminum; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D005459:Fluorides; C032311:aluminum fluoride