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, the 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 essential for the localization of ras p21 to the plasma membrane for their cellular functions including cell transformation in case of oncogenic ras p21. Here, we report the development of a rapid and convenient assay procedure for FTase using phosphocellulose paper which has a binding affinity for proteins. The FTase is assayed as the transfer of [3H]farnesyl group from [3H]FPP to the ras p21 at pH 7.4 and 37 degrees C in the presence of rat brain cytosol followed by the binding of radioactive farnesylated ras p21 to the phosphocellulose paper. The radioactivity associated with ras p21 bound to the phosphocellulose paper was determined by scintillation counting after soaking the paper in trichloroacetic acid and washing with distilled water. Utilizing [3H]FPP and recombinant Ha-ras p21 as substrates in the reaction, the FTase followed Michaelis-Menten kinetics with Km values of 1.0 and 7.69 microM for respectively [3H]FPP and recombinant Ha-ras p21. The method reported here has the advantages over the other published assay procedures 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.
D019883:Alkyl and Aryl Transferases; D000818:Animals; D001921:Brain / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D051231:Farnesyltranstransferase; D005374:Filtration; D007700:Kinetics; D008297:Male; D015689:Oncogene Protein p21(ras) / Q000378:metabolism*; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012717:Sesquiterpenes; D013379:Substrate Specificity; D014166:Transferases / Q000378:metabolism*; D014316:Tritium
D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D014316:Tritium; C004808:farnesyl pyrophosphate; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051231:Farnesyltranstransferase; D015689:Oncogene Protein p21(ras)