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In vitro reconstitution of activation of PLCepsilon by Ras and Rho GTPases.

Gandarillas, NL; Bunney, TD; Josephs, MB; Gierschik, P; Katan, M; (2009) In vitro reconstitution of activation of PLCepsilon by Ras and Rho GTPases. Methods Mol Biol , 462 pp. 379-389.

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Abstract

Phosphatidylinositol-specific phospholipase C (PLC) enzymes catalyze the hydrolysis of phophatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to diacylglycerol (DAG) and inositol 1,4,5-triphosphate [Ins(1,4,5)P3]. PLCepsilon is a recently discovered isoform that has been shown to be activated by members of the Ras and Rho families of guanosine trisphosphatases (GTPases) as well as subunits of heterotrimeric G-proteins. We describe a method for expressing a truncated PLCepsilon variant as an MBP fusion protein in E. coli. Subsequently, we describe the methodology necessary to reconstitute this protein with K-Ras-4B and RhoA GTPases and measure its activation.

Type: Article
Title: In vitro reconstitution of activation of PLCepsilon by Ras and Rho GTPases.
Location: United States
Keywords: Animals, Baculoviridae, Cell Line, Cell Membrane, Cell-Free System, Chromatography, Affinity, Chromatography, Gel, Enzyme Activation, Escherichia coli, Isoenzymes, Phosphoinositide Phospholipase C, Rats, Sequence Deletion, Solubility, ras Proteins, rho GTP-Binding Proteins
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology
URI: http://discovery.ucl.ac.uk/id/eprint/1326665
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