Fig. 2.

Incubation with trypsin reveals that Gsα with Thr204 mutated to Ala or Gln displays partial resistance to tryptic digestion in the absence of added guanine nucleotide or in the presence of GDP or GTP, as is seen in the wild-type Gsα only in the presence of nonhydrolyzable GTP analogues, in this instance GTPγS. Wild-type and Thr mutants of Gsα were synthesized in vitro in the presence of [³⁵S]methionine and incubated for 15 min at 25 °C in 20 μL of 20 mM Hepes-NaOH, pH 7.5 and 5 mM MgCl₂ with 10 μL/mL trypsin in the absence or presence of 200 μM GDP or GTP, followed by addition of 2 μL of 100 μg/mL trypsin and a second incubation for 15 min at 25 °C. The reactions were stopped by addition of 20 μL of Laemmli's sample buffer with 1% SDS. The samples were subjected to SDS-PAGE through a 4–12% polyacrylamide gel and visualized by autoradiography.