GTP-binding assays

GTP hydrolysis by Ras, which is considerably slow, has been analyzed, usually by single turn-over measurements. Ras is loaded with GTP by incubation of Ras (being in the GDP form after preparation) with a large excess of GTP in the presence of EDTA, which leads to faster dissociation of the nucleotide. After change of buffer and removal of excess nucleotide, aliquots are taken at certain time intervals and the nucleotide composition is analyzed with the help of filter binding assays or HPLC. Series of experiments with mutations in the neighbor-... 

There is a wealth of biochemical data on the activation of G proteins by receptors. Based on this very large body of evidence coming largely from experiments with cell membranes and reconstituted systems with the purified proteins, the now classical G protein cycle has been proposed (Bourne et at., 1990 Gilman, 1987). The core characteristic of this cycle is that the activated receptor causes GTP binding and subsequent dissociation of the heterotrimeric G protein into the active a. and fly subunits. FRET- and BRET-based assays have recendy been developed in order to investigate G protein activation in intact systems and also to assess whether G protein dissociation is indeed a requirement for G protein activation in the intact cell membrane. 

TABLE 1 Activity of Delta-Selective Ligands in Opioid Receptor Binding Assays and Delta GTP-y[35S] Assays... 

Purification of GTP binding and hydrolyzing activity from bovine brain led to the isolation of a G protein with an a subunit of Mr 39000, that is a substrate for PTX [73-75]. It co-purifies with another PTX substrate of much lower abundance, also an afiy heterotrimer, but with an a subunit of M, 41000. The G protein with a of 39 kDa was termed G and its a subunit a or a39. The subscript o was meant to denote other , to distinguish it from the PTX substrate with 04 . The functional role of G is unknown. The protein with composition a4l/3y was termed Gj, because the GTP S-activated a subunit inhibited adenylyl cyclase activity in Gs defective cyc cells [76]. Isolated G did not have this effect [75]. Thus, the cyc assay identifies a4 Py of bovine brain but not a39/3y as a possible Gj. 

Agonist potency, ED50 and Emax, measured in [35S]GTP[yS] binding assays in guinea pig caudate... 

Table 4 Pharmacological properties of MCL 450 (19) for the p, 5, and K opioid receptors in [35S] GTP /S binding assay...

The GTP-y-S binding assay, which provides an indirect measure of the ability of ligands to induce coupling of receptors to G-proteins. 

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