Detection of a 65 kDaras binding protein in rat and sheep brain cytosol using a chemical cross linking agent

The ability of a ras protein to associate with proteins present in rat brain cytosol in vitro was investigated using chemical cross-linking agents and the¹²⁵I-labelled v-H-ras protein. Two iodinated protein complexes with apparent molecular weights of 40 and 85 kDa were observed when a mixture of rat brain cytosol and [¹²⁵I]ras was treated with the cross-linking agent disuccinimidyl suberate and subjected to SDS-PAGE. Formation of the [¹²⁵I] 85 kDa complex was enhanced by a high concentration of EDTA while generation of the 40 kDa species was abolished by this treatment. Formation of the [¹²⁵I] 85 kDa complex was inhibited by unlabelled ras protein, GTP, GTPγS, and GDP but not by ATPγS and GMP. Chromatography of the cross-linked brain cytosol-[¹²⁵I]ras mixture on DEAE cellulose partially resolved the [¹²⁵I] 85 kDa complex from the [¹²⁵I]ras protein. The [¹²⁵I] 85 kDa complex (formed using ethyleneglycol bis (succinimidylsuccinate) as the cross-linking agent) could be immunoprecipitated using a rabbit anti-ras polyclonal antibody. Treatment of the immunoprecipitate with hydroxylamine to cleave the cross-link yielded [¹²⁵I]-labelled ras. A substantial enrichment of the proportion of the [¹²⁵I] 85 kDa complex in the cross-linked extract was achieved by preparative SDS-PAGE. It is concluded that the in vitro chemical cross-linking approach employed here has detected two ras binding proteins in rat brain cytosol: a 65 kDa heat-sensitive and a 20 kDa heat-stable protein. The possibility that the 65 kDa ras binding protein is a ras regulatory or ras effector protein which has not so far been characterised is briefly discussed.