Assay and isolation of palmitoyl-protein thioesterase from bovine brain using palmitoylated H-Ras as substrate

This chapter discusses the assay and isolation procedure of palmitoyl-protein thioesterase from bovine brain using palmitoylated H-Ras as substrate. In the case of H-Ras, palmitoylation increases association of the protein with cellular membranes and is required for full transforming activity. A dynamic cycle of Ras acylation and deacylation is demonstrated in vivo, suggesting a possible regulatory pathway mediating membrane association. Using H-Ras as a substrate, an assay for an enzyme that removes the covalently bound palmitate and purified it to homogeneity from a soluble fraction of bovine brain is developed. In addition to palmitoyl-Ras thioesterase activity, the enzyme has palmitoyl-CoA hydrolase activity comparable to mammalian thioesterases I and II and Escherichia coli thioesterase I; none of the other thioesterases removes palmitate groups from H-Ras. [³H]Palmitate-labeled, histidine-tagged H-Ras substrate is prepared using the insect baculovirus system. A recombinant baculovirus is created that encodes H-Ras with an N-terminal histidine affinity tag (vHis6-HRas) to facilitate protein purification. Infected Sf9 cells are metabolically labeled with [³H]palmitate, and [³H]palmitate-labeled, histidine-tagged H-Ras is purified from detergent-solubilized cell membranes by nickel affinity chromatography.