A RAPID AND CONVENIENT FILTER-BINDING ASSAY FOR RAS P21 PROCESSING ENZYME FARNESYLTRANSFERASE

Because it is the target for the development of anti-cancer agents, th e mammalian cytosolic enzyme farnesyltransferase (FTase) has received significant attention in recent years. FTase catalyzes the transfer of a farnesyl group from farnesylpyrophosphate (FPP) to cysteine(185/186 ) at the carboxyl terminal end of ras proteins (ras p21), a reaction e ssential for the localization of ras p21 to the plasma membrane for th eir cellular functions including cell transformation in case of oncoge nic ras p21. Here, we report the development of a rapid and convenient assay procedure for FTase using phosphocellulose paper which has a bi nding affinity for proteins. The FTase is assayed as the transfer of [ H-3]farnesyl group from [H-3]FPP to the ras p21 at pH 7.4 and 37 degre es C in the presence of rat brain cytosol followed by the binding of r adioactive farnesylated ras p21 to the phosphocellulose paper. The rad ioactivity associated with ras p21 bound to the phosphocellulose paper was determined by scintillation counting after soaking the paper in t richloroacetic acid and washing with distilled water. Utilizing [H-3]F PP and recombinant Ha-ras p21 as substrates in the reaction, the FTase followed Michaelis-Menten kinetics with K-m values of 1.0 and 7.69 mu M for respectively [H-3]FPP and recombinant Ha-ras p21. The method re ported here has the advantages over the other published assay procedur es of being rapid, convenient and economical, and can be successfully used for the basic assaying of FTase in different organs and distinct species and for the screening of novel inhibitors of FTase.