A stereospecific colorimetric assay for (S,S)-adenosylmethionine quantification based on thiopurine methyltransferase-catalyzed thiol methylation

被引:36
作者
Cannon, LM
Butler, FN
Wan, W
Zhou, ZS [1 ]
机构
[1] Washington State Univ, Dept Chem, Pullman, WA 99164 USA
[2] Washington State Univ, Sch Mol Biosci, Pullman, WA 99164 USA
关键词
S-adenosylmethionine; metabolite determination; colorimetric enzyme assays; stereospecificity; thiopurine S-methyltransferase; thiol methylation;
D O I
10.1016/S0003-2697(02)00267-1
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
S-Adenosyl-L-methionine (AdoMet) which is biologically synthesized by AdoMet synthetase bears an S configuration at the sulfur atom. The chiral sulfonium spontaneously racemizes to form a mixture of S and R isomers of AdoMet under physiological conditions or normal storage conditions. The chirality of AdoMet greatly affects its activity; the R isomer is not accepted as a substrate for Ado Met-dependent methyltransferases. We report a stereospecific colorimetric assay for (S,S)-adenosylmethionine quantification based on an enzyme-coupled reaction in which (S,S)-AdoMet reacts with 2-nitro-5-thiobenzoic acid to form AdoHcy and 2-nitro-5-methylthiobenzoic acid. The transformation is catalyzed by recombinant human thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) and is associated with a large spectral change at 410 nm. Accumulation of the S-adenosylhomocysteine (AdoHcy) product, a feedback inhibitor of TPMT, slows the assay. AdoHcy nucleosidase (EC 3.2.2.9) irreversibly cleaves AdoHcy to adenine and S-ribosylhomocysteine, significantly shortening the assay time to less than 10 min. The assay is linear from 5 to at least 60 muM (S,S)-AdoMet. (C) 2002 Elsevier Science (USA). All rights reserved.
引用
收藏
页码:358 / 363
页数:6
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