Figure 5.

Receptor-stimulated guanine nucleotide exchange. (Upper) Time course of binding of GTPγS to m1AChR-G_q vesicles. m1AChR-G_q proteoliposomes and 1 mM carbachol were incubated at 30°C for at least 3 min. Aliquots (12 fmol m1AChR, 47 fmol G_q) were diluted 1:2 with 500 nM [³⁵S]GTPγS for the times shown before quenching and measurement of bound [³⁵S]GTPγS. The solid line shows a fit to a first-order rate equation with k_exch = 0.135 s⁻¹. (Lower) Dependence of exchange rates on the concentration of free nucleotide. Nucleotide exchange was measured as described above in the presence of increasing concentrations of radiolabeled nucleotide at either 30°C (solid symbols) or 10°C (open symbols). Values of k_exch are plotted vs. the concentration of free nucleotide. At 10°C, exchange was relatively slow and was measured manually by using a protocol identical to that described for the quench-flow mixer except that the preincubation time was at least 15 min. Labeled nucleotides were [³⁵S]GTPγS in the presence of either 15 nM PLC-β1 (●, ○) or 4 μM RGS4 (⋄) or without GAP (■, □), or [α-³²P]GTP with PLC-β1 present (▴) or absent (▾).