DETECTION OF A 65-KDA RAS 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 crosslinking a gents and the I-125-labelled v-H-ras protein. Two iodinated protein co mplexes with apparent molecular weights of 40 and 85 kDa were observed when a mixture of rat brain cytosol and [I-125] ras was treated with the cross-linking agent disuccinimidyl suberate and subjected to SDS-P AGE. Formation of the [I-125] 85 kDa complex was enhanced by a high co ncentration of EDTA while generation of the 40 kDa species was abolish ed by this treatment. Formation of the [I-125] 85 kDa complex was inhi bited by unlabelled ras protein, GTP, GTPyS, and GDP but not by ATPyS and GMP. Chromatography of the cross-linked brain cytosol-[I-125] ras mixture on DEAF cellulose partially resolved the [I-125] 85 kD, comple x from the [I-125] ras protein. The [I-125] 85 kDa complex (formed usi ng ethyleneglycolbis (succinimidylsuccinate) as the cross-linking agen t) could be immunoprecipitated using a rabbit anti-ras polyclonal anti body. Treatment of the immunoprecipitate with hydroxylamine to cleave the cross-link yielded [(125)] I-labelled ras. A substantial enrichmen t of the proportion of the [I-125] 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 t wo 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.