Gbetagamma-copurified lipid kinase impurity from Sf9 cells.
G-protein betagamma dimers are prime regulators transmitting extracellular signals to wide-ranging cellular effectors including phosphoinositide 3-kinase (PI3K) isoforms beta and gamma. Recombinant Gbetagamma purified from Sf9 cells via metal-affinity and anion exchange chromatography exhibited a wortmannin-insensitive phospholipid kinase activity that copurified from the insect cells. To exclude false-positive results of Gbetagamma-dependent lipid kinase activity, the elimination of insect phospholipid kinase from Gbetagamma protein samples is necessary to avoid interference with the intrinsic lipid kinase activity of PI3K isoforms in reconstitution experiments. Here we describe an improved procedure of Gbeta(1)gamma(2) purification from cell membranes that separates the contaminating phospholipid kinase.
Shymanets
Aliaksei
A
Institut für Biochemie und Molekularbiologie II, Klinikum der Heinrich-Heine-Universität Düsseldorf, Klinikum der Eberhard-Karls-Universität Tübingen, Germany.
Ahmadian
Mohammad Reza
MR
Nürnberg
Bernd
B
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002850:Chromatography, Gel; D044387:GTP-Binding Protein beta Subunits / Q000302:isolation & purification* / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000302:isolation & purification* / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000302:isolation & purification / Q000378:metabolism*; D010770:Phosphotransferases / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification; D018411:Spodoptera / Q000378:metabolism; D000077191:Wortmannin
D000730:Androstadienes; C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D011994:Recombinant Proteins; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D000077191:Wortmannin