Real-time detection of basal and stimulated G protein GTPase activity using fluorescent GTP analogues

Hydrolysis of fluorescent GTP analogues BODIPY® FL guanosine 5′-O-(thiotriphosphate) (BGTPγS) and BODIPY® FL GTP (BGTP) by Gα_i1 and Gαo was characterized using on-line capillary electrophoresis laser-induced fluorescence assays in order that changes in substrate, substrate-enzyme complex, and product could be monitored separately. Apparent k_cat values (V_max/[E]) and K_m values were determined from steady-state assays for each substrate-protein pair. When BGTP was the substrate, maximum turnover numbers for Gα_o and Gα_i1 were 8.3 ± 1 × 10^-3 and 3.0 ± 0.2 × 10^-2 s^-1, respectively, and K _m values were 120 ± 60 and 940 ± 160 nM. Assays with BGTPγS yielded maximum turnover numbers of 1.6 ± 0.1 × 10^-4 and 5.5 ± 0.3 × 10^-4 s^-1 for Gα_o and Gα_i1; K_m values were 14 ± 8 and 87 ± 22 nM. Acceleration of Gα GTPase activity by regulators of G protein signaling (RGS) was demonstrated in both steady-state and pseudo-single-turnover assay formats with BGTP. Nanomolar RGS increased the rate of enzyme product formation (BODIPY® FL GDP (BGDP)) by 117-213% under steady-state conditions and accelerated the rate of G protein-BGTP complex decay by 199-778% in pseudo-single-turnover assays. Stimulation of GTPase activity by RGS proteins was inhibited 38-81% by 40 μM YJ34, a previously reported peptide RGS inhibitor. Taken together, these results illustrate that Gα subunits utilize BGTP as a substrate similarly to GTP, making BGTP a useful fluorescent indicator of G protein activity. The unexpected levels of BGTPγS hydrolysis detected suggest that caution should be used when interpreting data from fluorescence assays with this probe.