Rates of nucleotide exchange and GTP hydrolysis.

Nucleotide exchange was measured with intrinsic Trp fluorescence and with [35S]GTPγS binding. Rate of GTP hydrolysis was measured with single turn [γ-32P]GTP hydrolysis. The rate-limiting step was determined by comparing the rates of GTP binding and hydrolysis. The percentage of bound GTP was approximated by the following equation: GTP binding rate/(GTP binding rate + GTP hydrolysis rate). Note that GTP/GDP ratio and Gα interacting proteins will affect the state in vivo. Rates were measured at 20°C and are reported as min−1. *Fluorescence change was not detected, likely because of low specific activity of the recombinant protein. +not analyzed.