Figure 5.

Effect of mutations in the β2/β3 loop, α5 helix, and the β6/α5 loop on the GDP release rate of Gα_i1. The GDP release rate of wild type and mutant Gα_i1 subunit was measured using [³⁵S]GTPγS radioligand binding, as described.⁷⁸ GTPγS binding is an accurate method of measuring GDP release, given that GDP release is the rate limiting step in the nucleotide exchange process.¹¹ Data were fit to a single exponential function using GraphPad PRISM 3.0. Observed rate constants: (A) wild type, 0.036 min⁻¹; K192A, 0.056 min⁻¹; F336A, 0.1844 min⁻¹ (B) wild type, 0.068 min⁻¹; 0.16 min⁻¹, A326S. Note: the rate enhancement engendered by the A326S mutation in this experiment was only 3-fold, not the 20-fold previously reported.³² We observed faster GDP release by A326S Gα_i1 in some experiments. We hypothesize that this may be due to the idiosyncratic effects of polyoxyethylene 10-lauryl ether (lubrol) on GDP release, as described.¹²