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.
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 i subunits, possessed an approximate Mr of 42 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and copurified with a substoichiometric amount of beta subunit. Immunoblot analysis of fractions from the final Mono Q column revealed cross-reactivity of the 42-kDa phospholipase C activator with antipeptide antibodies raised against residues 160-169 of alpha i1 and a region of sequence common to all known G-protein alpha subunits. The 42-kDa activator was not recognized by other alpha subunit-specific or common antibodies. These findings identify the purified phospholipase C activator as a novel G-protein alpha subunit. This may represent the active subunit of the pertussis toxin-insensitive G-protein mediating receptor-stimulated phosphoinositide breakdown in mammalian liver.
Taylor
S J
SJ
Howard Hughes Medical Institute Laboratory, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295.
Smith
J A
JA
Exton
J H
JH
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D037342:Pertussis Toxin; D010738:Type C Phospholipases / Q000378:metabolism*; D014461:Ultracentrifugation; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D046911:Macromolecular Substances; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins