Interactions of the Ras-like Protein P25rab3A with Mg2+ and Guanine Nucleotides

The rab3A gene product is a 25 kDa guanine-nucleotide-binding protein which is expressed at high levels in neural tissue and has about 30% sequence similarity to ras. Purified p25rab3A has been used as substrate to examine its kinetics of nucleotide binding and hydrolysis, and to study the effects of Mg2+ on these processes. p25rab3A binds GDP and GTP similarly well, with nanomolar affinity. Mg2+ increases the affinity between p25rab3A and guanine nucleotides by 3- and 7-fold for GTP and GDP respectively, primarily by drastically decreasing the nucleotide off-rates. The Mg2+ binding affinity to p25rab3A. [alpha 32P]GDP was determined to be about 4 microns using entrapment of [alpha-32P]GDP as a measure of Mg2+ binding. At a Mg2+ concentration of 11 mM. GTPase activity was rate-limited by the GDP off-rate. Surprisingly, at a Mg2+ concentration of 80 nM. GTPase activity was comparable with that in the presence of excess Mg2+. In this case, kcat. was rate-limiting. At Mg2+ concentrations below 10 nM there was no detectable GTPase activity, indicating that Mg2+ is required for the GTPase activity of p25rab3A.