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[6] Quantitative reconstitution and ADP-ribosylation assays for G proteins

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DOI: 10.1016/s1043-9471(96)80042-5
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  • Biology

Abstract

Publisher Summary This chapter describes methods for the functional assessment and quantitation of the heterotrimeric G proteins that regulate adenylylcyclase activity, Gs and Gi. Two types of assays are described; one involves the reconstitution of Gs-stimulated adenylylcyclase activity in the cyc_variant of the S49 mouse lymphoma cell line and the other is the ability of the α subunits of these G proteins to be ADP-ribosylated by cholera and pertussis toxins. These assays are used to quantitate changes in Gs and Gi in corpora lutea after treatment of pseudopregnant rabbits with human chorionic gonadotropin and epinephrine. G proteins are responsible for conferring guanine nucleotide and Mg2+ sensitivity to receptors and effectors and for coupling agonist-occupied receptors to their effectors. The chapter discusses the development of functional reconstitution assays for Gs allowed for the characterization of the properties of Gs and is instrumental in the purification of Gs. These assays are modified so that they can be used to quantitate Gs functionally. Cholera and pertussis toxins have been useful tools in the identification and anaylsis of the structural and functional properties of Gs and Gi.

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