In vitro identification of a soluble protein:geranylgeranyl transferase from rat tissues

Abstract

The gamma subunit of mammalian trimeric G proteins has been shown previously to be modified in vivo on a cysteine residue situated at the carboxyl-terminal sequence-Cys-Ala-Ile-Leu-COOH by a 20-carbon prenyl moiety geranylgeranyl (Mumby, S. M., Casey, P. J., Gilman, A. G., Gutowski, S., and Sternweis, P. C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5873-5877; Yamane, H. K., Farnsworth, C. C., Xie, H., Howald, W., Fung, B. K-K., Clarke, S., Gelb, M. H., and Glomset, J. A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5866-5872). A biotinylated peptide acceptor comprising the eight carboxyl-terminal amino acids of the gamma subunit and tritiated geranylgeranyl diphosphate were utilized to monitor a protein:prenyl transferase activity in rat organs of varying age. The transferase activity was dependent upon the presence of divalent metal ions and maximal activity was achieved with either 1 mM ZnCl2 or 20 mM MgCl2. Activity was shown to be linear with respect to time, protein concentration, substrate concentration, and the pH optimum was 7.5. Protein:geranylgeranyl transferase activity was detected in all rat organs studied with the highest specific activity in brain S100. No activity was detected in the membrane fraction. The specific activity in brain, liver, kidney, and heart increased with age. Radioactivity incorporated into the peptide acceptor from both [1-3H]geranylgeranyl diphosphate and [5-3H]mevalonate by 21-day-old rat brain S100 was released by treatment with methyl iodide, and in both cases, analysis of the cleavage products by reversed phase high performance liquid chromatography showed a peak of radioactivity co-eluting with a geranylgeraniol standard which was well resolved from a farnesol standard. This indicated that the rat brain S100 contained not only the protein:geranylgeranyl transferase but also geranylgeranyl synthetase activity and that the peptide acceptor was specific for geranylgeranyl under the conditions tested.