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[4] G protein activity in solubilized membrane preparations

Methods in Neurosciences, ISSN: 1043-9471, Vol: 29, Issue: C, Page: 50-57
1996
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This chapter discusses the G protein activity in solubilized membrane preparations. The regulatory mechanisms require a cell-free approach that minimizes the potential contribution of indirect cellular compensatory mechanisms. A solubilized membrane system has been used to study G protein regulation of PLC activity. A nondenaturing detergent, such as sodium cholate, is used to extract membrane proteins in solution. Peripheral membrane proteins as well as trapped cytosolic proteins are also released in solution. If successful, the detergent-extracted membrane proteins retain functional activity and used for the study of interaction between PLC and G proteins in a detergent solution. Solubilization is often the first step in a purification protocol. The biochemistry of the system prior to final purification can provide insight as to whether the properties of the system have been affected by the purification scheme as might occur through the loss of a regulatory component. Using protocols similar to those originally developed for the solubilization of G proteins linked to adenylylcyclase, have shown that similar approaches result in the solubilization of a G-proteinregulated PLC. Thus, specific guanine nucleotide-dependent regulation of PLC activity is retained in the solubilized state. The following method for solubilization has been slightly modified from the original protocols to decrease the time needed to perform the entire procedure.

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