Purification and characterization of two G-proteins that activate the beta 1 isozyme of phosphoinositide-specific phospholipase C. Identification as members of the Gq class.
Plasma membranes from bovine liver contain a phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC) activity that is activated by guanine nucleotides. The G-proteins involved retained their ability to activate bovine brain PLC-beta 1 in a guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-dependent manner following extraction from the membranes with cholate and reconstitution with phospholipids. This reconstitution assay was used to purify the G-proteins by chromatography on heparin-Sepharose, DEAE-Sephacel, octyl-Sepharose, hydroxylapatite, Mono Q, and Sephacryl S-300 gel filtration. Gel electrophoresis showed that two alpha-subunits with molecular mass of 42 and 43 kDa were isolated to a high degree of purity, together with a beta-subunit. Neither alpha-subunit was a substrate for pertussis toxin-catalyzed ADP-ribosylation. Gel filtration of the final activity indicated an apparent molecular mass of 95 kDa, suggesting the presence of an alpha beta gamma heterotrimer. Immunological data revealed that the 42- and 43-kDa proteins were related to alpha-subunits of the Gq class recently purified from brain (Pang, I.-H., and Sternweis, P. C. (1990) J. Biol. Chem. 265, 18707-18712) and identified by molecular cloning (Strathmann, M., and Simon, M. I. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9113-9117). The activation of PLC-beta 1 by the purified G-protein preparation was specific for nonhydrolyzable guanine nucleotides, the efficacy decreasing in order GTP gamma S greater than guanylimidodiphosphate greater than guanylyl(beta,gamma-methylene)-diphosphonate. Half-maximal activation required 4 microM GTP gamma S suggesting that the affinity of the G-proteins for GTP analogues is low. The GTP gamma S-dependent activation of PLC-beta 1 required millimolar Mg2+ and was inhibited by guanosine 5'-O-(2-thiodiphosphate) and by excess beta gamma-subunits. Aluminum fluoride also activated PLC-beta 1 in the presence of the G-proteins. The G-proteins were inactive toward PLC-gamma 1 or PLC-delta 1. In summary, these findings identify two G-protein activators of PLC-beta 1 that have the properties of heterotrimeric G-proteins and are members of the Gq class.
Blank
J L
JL
Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295.
Ross
A H
AH
Exton
J H
JH
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002853:Chromatography, Liquid; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D008274:Magnesium / Q000378:metabolism; D037342:Pertussis Toxin; D010716:Phosphatidylinositols / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007527:Isoenzymes; D010716:Phosphatidylinositols; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D037342:Pertussis Toxin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D008274:Magnesium; D002118:Calcium