Journal
PROTEIN AND PEPTIDE LETTERS
Volume 16, Issue 9, Pages 1053-1056Publisher
BENTHAM SCIENCE PUBL LTD
DOI: 10.2174/092986609789055340
Keywords
Gel filtration; G-proteins; G beta gamma; phospholipid kinase; PI3K gamma; signal transduction; size exclusion chromatography; wortmannin
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Funding
- DFG (Deutsche Forschungsgemeinschaft)
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G-protein beta gamma dimers are prime regulators transmitting extracellular signals to wide-ranging cellular effectors including phosphoinositide 3-kinase (PI3K) isoforms beta and gamma. Recombinant G beta gamma 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 G beta gamma-dependent lipid kinase activity, the elimination of insect phospholipid kinase from G beta gamma 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 G beta(1)gamma(2) purification from cell membranes that separates the contaminating phospholipid kinase.
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