Regulation of purified subtypes of phosphatidylinositol-specific phospholipase C beta by G protein alpha and beta gamma subunits.
Specific antisera were produced to peptides representing the carboxyl termini of three subtypes of phosphatidylinositol-specific phospholipase C (PIPLC) beta which have been identified by isolation of cDNAs (Kriz, R., Lin, L., Sultzman, L., Ellis, C., Heldin, C., Pawson, T., and Knopf, J. (1990) Ciba Found. Symp. 150, 112-127). Screening with the antisera indicates that PIPLC beta 3 is present in a variety of cell lines and rat tissues, whereas the distribution of PIPLC beta 1 and beta 2 is more restricted. A combination of conventional and immunoaffinity chromatographic techniques was used to purify PIPLC beta 1 and beta 3 from rat brain membranes. PIPLC beta 2 was purified from cytosol of HL60 cells. All three subtypes were activated by purified G protein alpha q/11 subunits with the following relative efficacies: PIPLC beta 3 > or = PIPLC beta 1 >> PIPLC beta 2. All three PIPLC subtypes were also activated by G protein beta gamma subunits with varying efficacies. The presence of beta gamma subunits depressed the ability of alpha q/11 to activate PIPLC beta 1 and beta 3 at low Mg2+ concentrations (1 mM). At higher concentrations of Mg2+ (2 mM or greater), activation of PIPLC beta 3, but not PIPLC beta 1, by beta gamma and alpha q/11 became additive. PIPLC beta 3 was activated by alpha q/11 even in the presence of a saturating concentration of beta gamma subunits. This indicates that there are separate sites for interaction of PIPLCs with G protein subunits and that this interaction differs depending on the enzyme subtype and the concentration of Mg2+.
Smrcka
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Department of Pharmacology, Southwestern Graduate School of Biomedical Science, University of Texas Southwestern Medical Center, Dallas 75235-9041.
Sternweis
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D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D046911:Macromolecular Substances; D015636:Magnesium Chloride / Q000494:pharmacology; D008954:Models, Biological; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D051381:Rats; D014407:Tumor Cells, Cultured
D007074:Immunoglobulin G; D007527:Isoenzymes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D015636:Magnesium Chloride; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D019204:GTP-Binding Proteins; D043265:Phosphatidylinositol Diacylglycerol-Lyase