Characterization of a guanine nucleotide-releasing factor and a GTPase-activating protein that are specific for the ras-related protein p25rab3A.
The rab3A gene product is a 25-kilodalton guanine nucleotide-binding protein that is expressed at high levels in neural tissue and has about 30% homology to the ras gene product. Recombinant Rab3A protein and p25rab3A purified from bovine brain membranes have been used as substrates to look for factors that regulate its biochemical activity. A factor in rat brain cytosol exists that accelerates, by approximately 10-fold, the release and subsequent rebinding of guanine nucleotides to both native and recombinant p25rab3A. We have partially purified this activity, termed Rab3A-GRF, and a GTPase-activating protein (Rab3A-GAP) reported previously. The two activities copurified through a variety of procedures but were separated by Mono Q anion-exchange chromatography, indicating that the activities arise from distinct polypeptides. Both factors were thermolabile, sensitive to trypsin, and specific for Rab3A, exhibiting little or no activity toward c-Ha-Ras or Rab2 proteins. By gel filtration chromatography and sucrose density ultracentrifugation, both Rab3A-GRF and Rab3A-GAP have Stokes radii of 79 A and sedimentation coefficients of 8.9 S. We calculate a molecular mass of 295,000 daltons and a frictional ratio of 1.80 for each factor.
Burstein
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Department of Pathology, University of Vermont, Burlington 05405.
Macara
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D000818:Animals; D001923:Brain Chemistry; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000502:physiology*; D020690:GTPase-Activating Proteins; D006150:Guanine Nucleotides / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000502:physiology*; D011506:Proteins / Q000502:physiology*; D051381:Rats; D020692:rab3 GTP-Binding Proteins; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D006150:Guanine Nucleotides; D009419:Nerve Tissue Proteins; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D019204:GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins