Summary
The activity of mammalian phosphoinositide-specific phospholipase C β (PLCβ) is regulated by the αq family of G protein α subunits and by βγ subunits thought to be released from Gi. Interactions between G protein subunits and PLCβ can be assayed by measuring the stimulation of PLCβ enzymatic activity on reconstituting the purified G protein subunits with purified PLCβ on artificial phospholipid vesicles containing the substrate, phosphatidylinositol-4,5-bisphosphate (PIP2). These vesicles are doped with [3H]-inositol PIP2 and the rate of hydrolysis is determined by quantitating the amount of [3H]-inositol triphosphate (IP3) released from the vesicle into the aqueous phase. This assay provides a relatively simple method for assessing the activity PLC activity and its ability to be regulated by βγ and αq subunits. It can also be used to assess the functionality of the components after modification by mutagenesis, chemical modification, or in the presence of competing molecules.
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Ghosh, M., Smrcka, A.V. (2004). Assay for G Protein-Dependent Activation of Phospholipase C β Using Purified Protein Components. In: Smrcka, A.V. (eds) G Protein Signaling. Methods in Molecular Biology™, vol 237. Humana Press. https://doi.org/10.1385/1-59259-430-1:67
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DOI: https://doi.org/10.1385/1-59259-430-1:67
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