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 (Pi ) 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 Pi is directly proportional to the amount of [γ-32 P]GTP hydrolyzed, and, therefore, proportional to the activity of GTPases in the preparation.