UBC Theses and Dissertations
Guanine nucleotide binding properties and attempted immunopurification of ras protein from dictyostelium discoideum Bramble, Sharyl Elizabeth
One purpose of this study was to determine whether the ras protein from Dictyostelium discoideum (p23) binds guanine nucleotides like the ras proteins from mammals (p21) and yeast. The other purpose of this investigation was to purify or enrich for p23ras from D. discoideum by immunoaffinity chromatography. A number of different approaches were used to determine guanine nucleotide binding by p23RAS . A simple filter binding assay, binding to Western blots, and photoaffinity labeling all failed to demonstrate specific binding with lysates of D. discoideum cells. In contrast p21RAS from transformed NIH-3T3 cell lysate was successfully photoaffinity labeled in the presence of ³²P-α-guanosine 5¹-triphosphate (GTP) suggesting that the technique had been performed correctly. It was concluded that either p23RAS has a very low affinity for guanine nucleotides such that GTP binding was not detectable in these experiments or that the ras protein from D. discoideum simply does not bind guanine nucleotides. The purification of p23RAS from D. discoideum cells was attempted in order to provide a purified protein preparation for guanine nucleotide binding and for reconstitution studies. An anti-ras monoclonal antibody (Y13-259) was used as the ligand for the immunoaffinity chromatography. This approach was not successful in that the ras protein could not be enriched relative to other proteins because the immunoaffinity columns did not bind p23RAS.