Characterization and purification from bovine neutrophils of a soluble guanine-nucleotide-binding protein that mediates isozyme-specific stimulation of phospholipase C beta2.
Members of the beta isozyme subfamily of phosphatidylinositol-specific phospholipase C (PLC) are stimulated by alpha subunits and betagamma dimers of heterotrimeric guanine-nucleotide-binding proteins (G proteins). Myeloid differentiated human HL-60 granulocytes and bovine neutrophils contain a soluble phospholipase C, which is stimulated by the metabolically stable GTP analogue guanosine (5'-->O)-3-thiotriphosphate (GTP[S]). To identify the component(s) involved in mediating this stimulation, the relevant polypeptide(s) was resolved from endogenous phospholipase C and purified from bovine neutrophil cytosol by measuring its ability to confer GTP[S] stimulation to exogenous recombinant PLCbeta2. The resolved factor, which behaved as 48-kDa protein upon gel filtration, stimulated PLCbeta2 but not PLCbeta1 or PLCdelta1. Activation of phosphatidylinositol 4-phosphate 5-kinase was not involved in this stimulation. The purified stimulatory factor consisted of two polypeptides of molecular masses of approximately 23 kDa and 26 kDa. The protein stimulated a deletion mutant of PLCbeta2 that lacked a carboxyl-terminal region necessary for stimulation by members of the alpha(q) subfamily of the G-protein alpha subunits. The results of this study suggest that a GTP-binding protein distinct from alpha(q) subunits, probably a low-molecular-mass GTP-binding protein associated with a regulatory protein, is involved in isozyme-specific activation of PLCbeta2.
Illenberger
D
D
Department of Pharmacology and Toxicology, University of Ulm, Germany.
Schwald
F
F
Gierschik
P
P
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002850:Chromatography, Gel; D003600:Cytosol / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D009504:Neutrophils / Q000737:chemistry*; D009711:Nucleotides / Q000494:pharmacology; D054799:Phospholipase C beta; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
D007295:Inositol Phosphates; D007527:Isoenzymes; D008081:Liposomes; D009711:Nucleotides; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010738:Type C Phospholipases; C518036:PLCB2 protein, human; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins; D002118:Calcium