Partial purification of protein farnesyl cysteine carboxyl methyltransferase from bovine brain

• Published in: Experimental & molecular medicine 1998-12, Vol.30 (4), p.227-234
• Main Authors: Yoo, B C, Kang, M S, Kim, S, Lee, Y S, Choi, S Y, Ryu, C K, Park, G H, Han, J S
• Format: Article
• Language: English
• Subjects: Acetylcysteine - analogs & derivatives; Acetylcysteine - pharmacology; Animals; Brain - enzymology; Cattle; Chromatography, Liquid; Guanosine 5'-O-(3-Thiotriphosphate) - pharmacology; Molecular Weight; Protein Methyltransferases - chemistry; Protein Methyltransferases - isolation & purification; Protein Processing, Post-Translational
• ISSN: 1226-3613; 2092-6413; 2092-6413
• DOI: 10.1038/emm.1998.33

C-terminal farnesyl cysteine carboxyl methylation has been known to be the last step in the post-translational modification processes of several important signal transduction proteins in eukaryotes including ras related GTP binding proteins and the gamma-subunit of heterotrimeric G proteins. Protein farnesyl cysteine carboxyl methyltransferase (PFCCMT; EC, 2.1.1.100) catalyzing the reaction is well characterized as being stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and suppressed by N-acetyl-S-farnesyl-L-cysteine (AFC). As an initial step to understand the physiological significance of the process, we attempted to purify the enzyme, which was partially purified 130-fold (specific activity, 143 pmol of methyl group transferred/min/mg of protein) with yield of 1.8% after purification by fast protein liquid chromatography (FPLC) on a Superdex 75 column. The enzyme was further purified with non denaturing polyacrylamide gel electrophoresis (ND-PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of PFCCMT was determined to be about 30 kDa based on Superdex 75 FPLC as well as photoaffinity labelling with S-adenosyl-L-[methyl-3H] methionine ([methyl-3H]SAM). The partially purified enzyme (Superdex 75 eluate) was found to be characteristically affected by GTP gamma S, being activated about 40-fold in 2 mM, in contrast to ATP which did not show any effect on enzyme activity. Meanwhile, the enzyme was found to be markedly inhibited by AFC, reaching 0 activity in 2 mM. These observations strongly suggested that the partially purified enzyme was PFCCMT.