Partial purification of protein farnesyl cysteine carboxyl methyltransferase from bovine brain
Experimental & Molecular Medicine
;
: 227-234, 1998.
Artigo
em Inglês
| WPRIM
| ID: wpr-159765
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.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Proteínas Metiltransferases
/
Acetilcisteína
/
Encéfalo
/
Processamento de Proteína Pós-Traducional
/
Guanosina 5'-O-(3-Tiotrifosfato)
/
Cromatografia Líquida
/
Animais
/
Peso Molecular
Limite:
Animais
Idioma:
Inglês
Revista:
Experimental & Molecular Medicine
Ano de publicação:
1998
Tipo de documento:
Artigo
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