A comparison of proteins S-thiolated by glutathione to those arylated by acetaminophen

Raymond B. Birge, John B. Bartolone, Steven D. Cohen, Edward A. Khairallah, Lori A. Smolin

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28 Scopus citations

Abstract

This study was designed to evaluate whether the same proteins that irreversibly bind reactive electrophiles of drugs also bind glutathione (GSH) under oxidative conditions. Specifically, proteins that can be arylated by acetaminophen were compared to those that form glutathione-protein mixed disulfides (PSSG) after incubation with diamide. Data are presented which suggest that both GSH and acetaminophen bind to a subset of N-ethylmaleimide (NEM)-reactive protein thiols. To evaluate the pattern of proteins that bind GSH, PSSGs were formed in vitro by incubating cytosolic proteins with GSH and diamide. A sensitive procedure was developed in which PSSGs were first reduced with 0.1 mM dithiothreitol (DTT), and the newly exposed protein thiols were labeled with either [3H]NEM (for quantitative analysis) or with fluorescein-5-maleimide (for visual detection). Acetaminophen binding was achieved by incubating cytosolic proteins in vitro with the reactive acetaminophen metabolite, N-acetyl-P-benzoquinoneimine (NAPQI). Proteins from both assays were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose for Western blot analysis. Acetaminophen binding was detected by immunoblotting with an affinity-purified antibody against acetaminophen, and PSSGs were visualized using anti-fluorescein antibodies. In both instances, binding to proteins was observed to be selective. A comparison of the proteins modified by GSH binding with those that bind acetaminophen indicates that the major cytosolic acetaminophen-binding protein of 58 kDa may also be modified by glutathiolation under oxidative conditions.

Original languageEnglish (US)
Pages (from-to)S197-S207
JournalBiochemical Pharmacology
Volume42
Issue numberSUPPL. 1
DOIs
StatePublished - Dec 11 1991
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Pharmacology

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