Partial purification of protein farnesyl cysteine carboxyl methyltransferase from bovine brain.
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.
Yoo
B C
BC
Department of Biochemistry, Medical College, Korea University, Seoul, Korea.
Kang
M S
MS
Kim
S
S
Lee
Y S
YS
Choi
S Y
SY
Ryu
C K
CK
Park
G H
GH
Han
J S
JS
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002853:Chromatography, Liquid; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D008970:Molecular Weight; D011496:Protein Methyltransferases / Q000737:chemistry / Q000302:isolation & purification*; D011499:Protein Processing, Post-Translational
D016244:Guanosine 5'-O-(3-Thiotriphosphate); D011496:Protein Methyltransferases; C056501:protein-S-isoprenylcysteine O-methyltransferase; C068268:N-acetyl-S-farnesylcysteine; D000111:Acetylcysteine