Hyperplasia in the rabbit bladder urothelium following partial outlet obstruction. Autoradiographic evidence

Recombinant histidine-tagged v-Ha-ras (his-ras) was purified to homogeneity from extracts ofE. coli M15 using a one-step procedure which involved immobilised metal ion chromatography on Ni²⁺-nitriloacetic acid agarose (Ni-NTA). The optimal pH for elution by imidazole was 6.6 and the yield of his-ras protein (greater than 95% pure) was about 4 mg/litreE. coli culture. Chromatography of a mixture of purified his-ras and rat brain cytosol on Ni-NTA together with SDS-PAGE and silver staining of proteins were employed to search forras-binding proteins present in rat brain cytosol. Chromatography of rat brain cytosol alone on Ni-NTA revealed several protein species which were not readily eluted with imidazole. These are likely to be low-abundance brain metal ion binding proteins. Pre-treatment of rat brain cytosol with Ni-NTA before a second round of chromatography on Ni-NTA removed most of these proteins. Chromatography of a mixture of pre-treated rat brain cytosol and purified his-ras protein revealed four new protein bands with molecular weights of 250, 90, 80 and 70 kDa. These were considered to be candidateras-binding proteins. It is concluded that the use of his-ras and immobilised metal ion chromatography does provide an approach which can be used to identifyras binding proteins present in cellular extracts.Abbreviations his-ras histidine-tagged vHa-ras - Ni-NTA Ni²⁺ nitriloacetic acid agarose - IPTG isopropyl thio--D-galactoside