8] Measurement of intrinsic nucleotide exchange and GTP hydrolysis rates

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This chapter describes methods to measure nucleotide exchange rates and GTP hydrolysis rates of Rho, Rac, and G25K. Like all GTP-binding proteins, Rho-related GTPases exist in two conformational states: an inactive GDP-bound form and an active GTP-bound form; their interconversion occurs through a cycle of guanine nucleotide exchange and GTP hydrolysis. The intrinsic nucleotide exchange and GTPase activities of all members of the Ras superfamily are relatively slow, and in vivo, they are stimulated by guanine exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs and GAPs for the members of the Rho subfamily have been identified, although their cellular roles and exact specificities are still unclear. The first GAP described for this family (Rho–GAP) was purified biochemically from human spleen and was shown to be active on Rho, but it is also active on Rac and CDC42/G25K. Another important regulator of the Rho family of proteins is the Rho–GDP-dissociation inhibitor (GDI), Rho–GDI. Guanine nucleotide off rates are discussed in the chapter.