Measurement of the GTPase Activity of Signal-Transducing G-Proteins in Neuronal Membranes

The methods described in this chapter are designed to measure the hydrolysis of guanosine triphosphate (GTP) to inorganic phosphate (P_i ) and guanosine diphosphate (GDP), a reaction catalyzed by the GTPase enzymes (EC 3.6.1.-). The theory behind the experimental design involves using [γ-³² P]GTP as a marker, whereby any GTPase-induced hydrolysis will result in the ³² P label appearing as ³² P, and as unhydrolyzed [γ-³² P]GTP. It was originally described by Cassel and Selinger (1 ). ³² P, must be separated from [γ-³² P]GTP, and then can be easily quantitated by using a liquid scintillation counter. The amount of ³² P_i is directly proportional to the amount of [γ-³² P]GTP hydrolyzed, and, therefore, proportional to the activity of GTPases in the preparation.