A stereospecific colorimetric assay for (S,S)-adenosylmethionine quantification based on thiopurine methyltransferase-catalyzed thiol methylation
Section snippets
Materials
AdoMet p-toluenesulfonate salt and ATP disodium salt were purchased from Sigma. 5,5′-Dithiobis-2-nitrobenzoic acid (DTNB; Ellman's reagent) was purchased from Aldrich. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was purchased from Fluka. l-Methionine, isopropyl-β-d-thiogalactoside (IPTG), dithiothreitol (DTT), and kanamycin were purchased from Fisher. HiTrap Chelating HP columns were purchased from Amersham Pharmacia. All other chemicals were of the highest purity available.
Preparation of reagents
A solution of
Results and discussion
Ames and co-workers [21] reported that human thiopurine S-methyltransferase methylates a series of aromatic thiols, including 4-nitrothiophenol, using AdoMet as the methyl donor. Since methylation of 4-nitrothiophenol is associated with a significant UV–visible spectral change, we reasoned that a TPMT-catalyzed thiol methylation could be used to quantify AdoMet. From 1 L of growth medium, about 100 mg recombinant human TPMT was purified to homogeneity as judged by Coomassie-stained SDS–PAGE gel.
Acknowledgements
We are indebted to Dr. Richard Weinshilboum at Mayo Clinics for providing human TPMT cDNA, Dr. G. Doug Markham at Fox Chase Cancer Center for providing AdoMet synthetase expression strain and purified enzyme, and Drs. Kenneth A. Cornell and Michael K. Riscoe at the Portland VA Medical Center for providing AdoHcy nucleosidase expression strain and purified enzyme. We also thank Drs. Cornell and Markham for comments and suggestions; L.M.C. and F.N.B. were supported by a mini-grant from the
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