Figure 6. PMT inhibits the GTPase activity of G_q.

(A) Membrane proteins were incubated in the presence or absence of 150 pM PMT for 1 h with 0.5 nM GTP and tested in a trypsin protection assay as described in Materials and Methods. Proteins were separated by SDS PAGE and Western blotted with anti-Gα_q/11 antibody. Quantification of activated Gα_q/11 (lower panel) was determined by densitometric scanning and the data were analysed using factorial analysis of variance (ANOVA). The induction of activation shown is relative to the density of the band without GTP or PMT. PMT significantly enhanced GTP binding to G_q (* p = 0.03). (B) Membrane proteins were incubated in the presence or absence of 30 nM bombesin 20 min with 0.5 nM GTP and tested in a trypsin protection assay as described in Materials and Methods. Proteins were separated by SDS PAGE and Western blotted with anti-Gα_q/11 antibody. Samples from at least 3 independent membrane preparations were resolved with similar results. (C) Membranes derived from Swiss 3T3 cells that had either been treated or untreated with 150 pM PMT for 4 h or 100 ng cholera toxin, or both, were treated with or without 30 nM bombesin for 20 min in the presence of [γ-³²P] GTP. All the experimental conditions were repeated three times, and all data are presented as mean ± standard deviation (SEM). The results for the groups were compared using single-factor analysis of variance (one-way ANOVA), followed by Newnan-Keuls test used to determine differences between groups. Significant changes are indicated by an asterisk (* P<0.05, *** P<0.001). (D) Membranes derived from Swiss 3T3 cells that had either been untreated (lane 1) or treated with 150 pM PMT for 4 h (lane 2), or 100 ng cholera toxin for 16 h (lane 3), or both PMT and cholera toxin (lane 4) were resolved by SDS PAGE followed by Western blotting with an anti-Gα_s antibody. Samples from at least 3 independent membrane preparations were resolved with similar results.