Journal: Plant Signaling & Behavior

Article Title: A sweet cycle for Arabidopsis G-proteins

doi:

Figure Lengend Snippet: Effect of mutations in the β2/β3 loop, α5 helix, and the β6/α5 loop on the GDP release rate of Gαi1. The GDP release rate of wild type and mutant Gαi1 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 Gαi1 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

Article Snippet: 11 Data were fit to a single exponential function using GraphPad PRISM 3.0.

Techniques: Mutagenesis, Binding Assay

Journal: Plant Signaling & Behavior

Article Title: A sweet cycle for Arabidopsis G-proteins

doi:

Figure Lengend Snippet: Saturation binding analysis of the affinity of Mg2+·GTPγS for AtGPA1. 1 nM AtGPA1 was mixed with various concentrations of [35S] GTPγS in the presence of 25 mM MgCl2.6,78 Bound GTPγS was quantified by filtration and liquid scintillation as described.6,78 Non-specific binding was determined in the presence of 100 mM unlabeled GTPγS. Specific binding was fit to a saturation binding isotherm (Y = Bmax + X / (KD + X)) using GraphPad PRISM 3.0.

Article Snippet: 11 Data were fit to a single exponential function using GraphPad PRISM 3.0.

Techniques: Binding Assay, Filtration