Characterization of human platelet GTPase activating protein for the Ral GTP-binding protein.
RalA, a ras p21 related 27 kDa GTP-binding protein, was expressed as a fusion protein in Escherichia coli and purified to homogeneity using an immunoaffinity column. The purified protein was capable of binding and hydrolyzing GTP. Addition of platelet cytosolic or detergent solubilized particulate proteins stimulated the intrinsic GTPase activity of ralA by at least six-fold with maximal effect observed at pH 6.5. Addition of platelet proteins denatured by boiling had no effect on ralA GTPase activity. Analysis of GTPase reaction products by thin layer chromatography demonstrated that in samples containing ralA, 78.5 +/- 6.3% of the radioactivity was recovered in the GTP form while samples containing ralA plus platelet cytosol or particulate proteins, only 7.5 +/- 0.2% and 9.0 +/- 1.4% of the radioactivity was in the GTP form respectively. The GTPase activating protein(s) in the cytosolic and particulate fraction was further characterized by measuring GAP activity in proteins eluted from gel slices after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The ralA GTPase activating protein present in the cytosol and particulate fractions was recovered in a single gel slice of identical apparent molecular weight. The molecular mass of the ral specific GTPase activating protein was estimated to be 34 +/- 2 kDa. This protein did not stimulate the intrinsic GTPase activity of ras p21, G25K/CDC42Hs or rab3A GTP-binding proteins. Results demonstrate that in human platelets, the activity/function of ral-related GTP-binding protein(s) is under the regulation of a specific GTPase activating protein of molecular mass of 34 +/- 2 kDa that is distributed equally in the cytosol and particulate fraction.
Bhullar
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RP
Department of Oral Biology, University of Manitoba, Winnipeg, Canada.
Seneviratne
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HD
D001792:Blood Platelets / Q000378:metabolism*; D002846:Chromatography, Affinity; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D015870:Gene Expression; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020725:ral GTP-Binding Proteins; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D020703:ras GTPase-Activating Proteins; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020725:ral GTP-Binding Proteins