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-32P]GTP as a marker, whereby any GTPase-induced hydrolysis will result in the 32P label appearing as 32P, and as unhydrolyzed [γ-32P]GTP. It was originally described by Cassel and Selinger (1). 32P, must be separated from [γ-32P]GTP, and then can be easily quantitated by using a liquid scintillation counter. The amount of 32Pi is directly proportional to the amount of [γ-32P]GTP hydrolyzed, and, therefore, proportional to the activity of GTPases in the preparation.
KeywordsGTPase Activity Guanosine Triphosphate Guanosine Diphosphate Trypsin Inhibitor Unit Nonhydrolyzable Analog
- 1.Cassel, D. and Selinger, Z. (1976) Catecholamine-stimulated GTPase activity in turkey erythrocyte membranes. Biochim. Biophys. Acta 452, 538–551.Google Scholar
- 4.Gierschik, P. and Jakobs, K. H. (1990) Receptor-stimulated GTPase activity of G-proteins, in G-Proteins as Mediators of Cellular Signalling Processes (Houslay, M. D. and Millgan, G., eds.), Wiley, London, pp. 67–82.Google Scholar
- 12.Sweeney, M. I. and Dolphm, A. C. (1993) Adenosine A1 agonists stimulate the GTPase activity of Gi-and Go-type G-proteins in cortical membranes: synergism with Bay K 8644. J. Neurochem. 61(Suppl.), S78D.Google Scholar