Abstract
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.
Highlights
GTP-binding regulatory proteins (G proteins) belong to a family that are present in all eukaryotic organisms from yeast to human
DEAE-cellulose was the product of Whatman Inc. (Clifton, U.S.A.). [methyl-3H]SAM was purchased from Amersham Co. (Buckinghamshire, UK), and 3[(3-cholamidopropyl)-dimethylammonio]-l-propanesulfonate (CHAPS) and guanosine 5'-O-(3-thiotriphosphate) (GTPγS) were obtained from Sigma
Partial purification of Protein farnesyl cysteine carboxyl methyltransferase (PFCCMT) and determination of molecular weight PFCCMT was partially purified from bovine brain following the methytransferase activity stimulated by GTPγS and inhibited by AFC
Summary
GTP-binding regulatory proteins (G proteins) belong to a family that are present in all eukaryotic organisms from yeast to human. Similarities in cellular location, structure, and biochemistry among them suggest that they play a role in transduction of signals from the cell (Schaefer et al, 1990). Included in this family are two classes of the G protein superfamily, the ras-related GTP-binding proteins and the γ-subunit of heterotrimeric G proteins (Pillinger et al, 1994). The biochemical nature of their final product in signal transduction is not fully understood, with respect to the maturation and processing of translational products. Since mutations should result in alterations in the subsequent transcription and translation and the final maturation processing reactions, it is highly important to understand the enzymology and regulation of post-translational modification reactions (Kato et al, 1992)
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