Purification of a 110-kDa phosphoinositide phospholipase C that is activated by G-protein beta gamma-subunits.
We report the purification from bovine brain cytosol of a 110-kDa phosphoinositide-specific phospholipase C (PLC-110) that was markedly stimulated by G-protein beta gamma-subunits. The enzyme was purified approximately 2000-fold with a yield of 4%. On the basis of size and immunological cross-reactivity, PLC-110 was distinct from 150-kDa PLC-beta 1, 145-kDa PLC-gamma 1, and 85-kDa PLC-delta 1. An antiserum to a peptide corresponding to a conserved PLC Y domain sequence cross-reacted with PLC-110. PLC-110 was also recognized by two antisera selective for NH2-terminal and internal sequences in PLC-beta 3, but not by a third peptide antiserum to the COOH terminus of this enzyme, suggesting that PLC-110 is related to PLC-beta 3. Reconstitution of purified PLC-110 with beta gamma-subunits produced greater than 100-fold activation, indicating activation was observed at approximately 60 nM beta gamma and full activation at approximately 500 nM beta gamma. PLC-110 maximally hydrolyzed phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate at 1 microM Ca2+, but showed no activity toward phosphatidylinositol at Ca2+ concentrations up to 1 mM. Concentrations of purified guanosine 5'-O-(3-thiotriphosphate)-liganded alpha q that fully activated PLC-beta 1 failed to stimulate PLC-110. This observation indicates that the site at which beta gamma interacts with PLC-110 is distinct from that at which alpha q regulates the activity of PLC-beta isozymes.
Blank
J L
JL
Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295.
Shaw
K
K
Ross
A H
AH
Exton
J H
JH
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003429:Cross Reactions; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010727:Phosphoric Diester Hydrolases; D019204:GTP-Binding Proteins; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002118:Calcium