title: Purification and G protein subunit regulation of a phospholipase C-beta from Xenopus laevis oocytes.
abstract: Xenopus oocytes exhibit both pertussis toxin-sensitive and -insensitive inositol lipid signaling responses to G protein-coupled receptor activation. The G protein subunits Galphai, Galphao, Galphaq, Galphas, and Gbetagamma all have been proposed to function as activators of phospholipase C in oocytes. Ma et al. (Ma, H.-W., Blitzer, R. D., Healy, E. C., Premont, R. T., Landau, E. M., and Iyengar, R. J. Biol. Chem. 268, 19915-19918) cloned a Xenopus phospholipase C (PLC-betaX) that exhibits homology to the PLC-beta class of isoenzymes. Although this enzyme was proposed to function as a signaling protein in the pertussis toxin-sensitive inositol lipid signaling pathway of oocytes, its regulation by G protein subunits has not been directly assessed. As such we have utilized baculovirus-promoted overexpression of PLC-betaX in Sf9 insect cells and have purified a recombinant 150-kDa isoenzyme. PLC-betaX catalyzes hydrolysis of phosphatidylinositol(4,5)bisphosphate and phosphatidylinositol(4)monophosphate, and reaction velocity is dependent on Ca2+. Recombinant PLC-betaX was activated by both Galphaq and Gbetagamma. PLC-betaX exhibited a higher apparent affinity for Galphaq than Gbetagamma, and Galphaq was more efficacious than Gbetagamma at lower concentrations of PLC-betaX. Relative to other PLC-beta isoenzymes, PLC-betaX was less sensitive to stimulation by Galphaq than PLC-beta1 but similar to PLC-beta2 and PLC-betaT. PLC-betaX was more sensitive to stimulation by Gbetagamma than PLC-beta1 but less sensitive than PLC-beta2 and PLC-betaT. In contrast PLC-betaX was not activated by the pertussis toxin substrate G proteins Galphai1, Galphai2, Galphai3, or Galphao. These results are consistent with the idea that PLC-betaX is regulated by alpha-subunits of the Gq family and by Gbetagamma and do not support the idea that alpha-subunits of pertussis toxin-sensitive G proteins are directly involved in regulation of this protein.
authors.affiliation: Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA. tkh@med.unc.edu
authors.forename: T M
authors.initials: TM
authors.lastname: Filtz
authors.forename: A
authors.initials: A
authors.lastname: Paterson
authors.forename: T K
authors.initials: TK
authors.lastname: Harden
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009865:Oocytes / Q000201:enzymology*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D054799:Phospholipase C beta; D010738:Type C Phospholipases / Q000302:isolation & purification* / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D007527:Isoenzymes; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; C037178:phosphatidylinositol 4-phosphate; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins; D002118:Calcium