SpringerProtocols: Abstract: 5. Assay for G Protein-Dependent Activation of Phospholipase C β Using Purified Protein Components

5. Assay for G Protein-Dependent Activation of Phospholipase C β Using Purified Protein Components

By: Mousumi Ghosh², Alan V. Smrcka²

Abstract

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The activity of mammalian phosphoinositide-specific phospholipase C β (PLCβ) is regulated by the α_q family of G protein α subunits and by βγ subunits thought to be released from G_i. 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 α_q 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.

Affiliation(s): (2) Department of Pharmacology and Physiology, University of Rochester School of Medicine, Rochester, NY

Book Title: G Protein Signaling: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 237 | Pub. Date: Sep-15-2003 | Page Range: 67-75 | DOI: 10.1385/1-59259-430-1:67

Subject: Protein Science

Key Words: Phospholipase regulation - PLCβ - G protein - Gαq - Gβγ

References

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