Calnuc does not affect binding of GTPγS to Gαi3. Addition of calnuc to Gαi3 has no effect on its rate of GDP exchange and binding of GTPγS, whereas mastoparan (used as a positive control) greatly enhances binding of GTPγS to Gαi3. Results (means ± SD) represent the average of values obtained in 3 separate experiments after subtraction of the blank (0.1% of the total radioactivity added). Mastoparan (100 μM) or His6-calnuc (1 μM) was incubated with 2 μM [35S]GTPγS. Reactions were started by adding 200 nM His6-Gαi3 and incubated at 30°C for 0–60 min. 50 μl of the reaction mixture were collected on nitrocellulose filters by vacuum filtration. Bound radioactivity was determined by liquid scintillation counting.