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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Baculoviridae / enzymology*
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Cell Line
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Cell Membrane / chemistry
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Cell-Free System
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Chromatography, Affinity
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Chromatography, Gel
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Enzyme Activation / drug effects
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Escherichia coli / cytology
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Isoenzymes / genetics
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Isoenzymes / isolation & purification
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Isoenzymes / metabolism
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Phosphoinositide Phospholipase C / genetics
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Phosphoinositide Phospholipase C / isolation & purification*
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Phosphoinositide Phospholipase C / metabolism*
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Rats
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Sequence Deletion
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Solubility
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ras Proteins / isolation & purification*
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ras Proteins / pharmacology*
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rho GTP-Binding Proteins / isolation & purification*
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rho GTP-Binding Proteins / pharmacology*
Substances
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Isoenzymes
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Phosphoinositide Phospholipase C
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ras Proteins
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rho GTP-Binding Proteins