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

L. A. Camp, S. L. Hofmann

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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.

Original languageEnglish (US)
Pages (from-to)336-347
Number of pages12
JournalMethods in Enzymology
Issue numberC
StatePublished - Jan 1 1995


ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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