Table 2. Rates of GTPγS binding and GTP hydrolysis in wild-type and chimeric Gα subunits.
Rates of GTPγS binding were determined by in vitro [35S]GTPγS binding assays. Rates of GTP binding were determined by intrinsic fluorescence measurements. Rates of GTP hydrolysis were measured by [γ-32P]GTP hydrolysis assay. The values of GTPγS binding and GTP hydrolysis (single-turnover) are shown as min−1. The steady-state hydrolysis values are given as GTP hydrolyzed/(min × Gα), where the amount of active Gα was determined by its ability to bind to GTPγS. Experiments for which no data were obtained are marked with an “x.”
| HsGαi1 | HsGαi1α5 hel | TvGα5αi1 hel | TvGα5 | |
|---|---|---|---|---|
| GTPγS binding | 6.4 × 10−3 ± 3.1 × 10−4 | 0.12 ± 0.03 | x | 0.80 ± 0.17 |
| Intrinsic fluorescence binding | 8.2 × 10−3 ± 5.2 × 10−3 | 0.25 ± 0.01 | 0.37 ± 0.02 | 6.53 ± 0.68 |
| GTP hydrolysis (single-turnover) | 0.82 ± 0.16 | 0.49 ± 0.05 | 0.0052 ± 0.001 | 6.2 × 10−3 ± 1.0 × 10−3 |
| Steady-state hydrolysis | 3.9 × 10−3 ± 5.8 × 10−4 | 0.41 ± 0.03 | 0.013 ± 0.002 | 6.1 × 10−3 ± 5.8 × 10−4 |
| Rate-limiting step* | Binding | Binding | Hydrolysis | Hydrolysis |
The rate-limiting step was found by comparing the rate of single-turnover (or estimated single-turnover) GTP hydrolysis to the rate of GTPγS binding. The rates shown in this table were computed from more than 12 data points of at least two experiments. Variability is represented by the SEM.