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.
Burstein
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Department of Pathology, University of Vermont, Burlington 05405.
Macara
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D000818:Animals; D001921:Brain / Q000378:metabolism; D002413:Cations, Divalent; D002417:Cattle; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D020692:rab3 GTP-Binding Proteins
D002413:Cations, Divalent; D006150:Guanine Nucleotides; D009419:Nerve Tissue Proteins; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D008274:Magnesium