Reconstitution of a solubilized membrane but not cytosolic phospholipase C with membrane-associated Gp from GH3 cells.
Hormones have been demonstrated to activate phosphoinositide hydrolysis in plasma membranes in a manner dependent upon or potentiated by GTP. For thyrotropin-releasing hormone activation in GH3 cell membranes, stimulation persisted in membranes from pertussis toxin-treated cells. These observations indicate the presence of a membrane phospholipase C (PL C) and a novel GTP-binding protein (Gp); however, neither of these proteins has been characterized. In this paper, we report studies of GH3 membrane PL C utilizing [3H]phosphatidylinositol 4,5-bisphosphate liposome substrate. Guanosine 5'-O-(3-thiotriphosphate) (GTP[S]), but not other nucleotides, was found to stimulate PL C activity and required greater than 1 nM Ca2+. High concentrations of Ca2+ (10 microM) also activated the membrane PL C. Treatment of membranes with N-ethylmaleimide inhibited Ca2+-activated but not GTP[S]-activated PL C. Extraction of membranes with 1 M KCl solubilized the membrane PL C; however, the solubilized PL C was not GTP[S]-stimulated. N-ethylmaleimide-treated, KCl-extracted membranes were markedly deficient in GTP[S]-stimulated PL C activity; however, activity could be restored by incubation with the desalted extracted PL C. Reconstitution appeared to involve the recoupling of membrane-associated Gp with soluble 330- and 110-kDa forms of the PL C. Cytosolic PL Cs failed to substitute for the solubilized membrane PL C. These results indicate that the Gp-regulated PL C in GH3 cell membranes is an extrinsic membrane protein that can be extracted reversibly at high ionic strength. Moreover, the membrane PL C can be distinguished from cytosolic PL C isoenzymes.
Martin
T F
TF
Department of Zoology, University of Wisconsin, Madison 53706.
Kowalchyk
J A
JA
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D005033:Ethylmaleimide / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology; D007700:Kinetics; D012995:Solubility; D010738:Type C Phospholipases / Q000302:isolation & purification / Q000378:metabolism*
D006150:Guanine Nucleotides; D000255:Adenosine Triphosphate; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D005033:Ethylmaleimide; D002118:Calcium