1.Partial purification of protein farnesyl cysteine carboxyl methyltransferase from bovine brain.
Byung Cheol YOO ; Myung Seo KANG ; Sang Duk KIM ; Young Sun LEE ; Soo Yeon CHOI ; Chong Keun RYU ; Gil Hong PARK ; Jong Seol HAN
Experimental & Molecular Medicine 1998;30(4):227-234
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.
Acetylcysteine/pharmacology
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Acetylcysteine/analogs & derivatives
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Animal
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Brain/enzymology*
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Cattle
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Chromatography, Liquid
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Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology
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Molecular Weight
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Protein Methyltransferases/isolation & purification*
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Protein Methyltransferases/chemistry
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Protein Processing, Post-Translational