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. These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.