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소의 뇌로부터 Protein Farnesyl-Cysteine Carboxyl-Methyltransferase의 정제에 관한 연구
강명서,박길홍,황우익 고려대학교 의과대학 1996 고려대 의대 잡지 Vol.33 No.1
Protein farnesyl-cysteine carboxyl-methyltransferse (PFCCMT) has been known to be involved in posttranslational modification of several important signal transduction proteins in eukaryotes including ras superfamily, γ-subunit of heterotrimeric G protein, α-subunit of cGMP phosphodiesterase isolated from retinal rod, yeast mating pheromone, which raises the possibility that carboxyl methylation may play a central role in the regulation of stimulus-response coupling in eukaryotic cells. The enzyme has been purified from bovine brain membranes, utilizing a synthetic substrate, N-acetyl-S-farnesyl-L-cysteine (AFC) , as the methyl acceptor substrate. The membrane-bound enzyme was found to be most effectively extracted from bovine brain with the buffer containing 0.6% 3 [3-cholamidopropyl) -dimethylammonio) -1-propane- sulfonate(CHAPS). The solubilized enzyme was purified by Fast Protein Liquid Chromatography (FPLC) employing Superose 6 and Mono Q columns, and non-denaturing polyacrylamide gel electrophoresis(ND-PAGE) 115 fold with the final specific activity of 496 pmol of [methyl-^(14)C] group trasfer/min/mg protein, and single band could be visualized on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme appeared to be 76,000 dalton on NDPAGE and 38,000 dalton on SDS-PAGE, indicating that it is composed of two identical subunits. Additionally, AFC was found to act as inhibitor in high concentrations above 0.1 mM, and the almost same enzyme activity measurements with and without AFC intriguingly suggests that the enzyme exists in complex strongly bound to in vivo substrates in the cell.