Metallothionein disulfides are present in metallothionein-overexpressing transgenic mouse heart and increase under conditions of oxidative stress

Wenke Feng, Frederick W. Benz, Jian Cai, William M. Pierce, Yujian Kang

Research output: Contribution to journalArticle

84 Citations (Scopus)

Abstract

Metallothionein (MT) releases zinc under oxidative stress conditions in cultured cells. The change in the MT molecule after zinc release in vivo is unknown although in vitro studies have identified MT disulfide bond formation. The present study was undertaken to test the hypothesis that MT disulfide bond formation occurs in vivo. A cardiac-specific MT-overexpressing transgenic mouse model was used. Mice were administered saline as a control or doxorubicin (20 mg/kg), which is an effective anticancer drug but with severe cardiac toxicity at least partially because of the generation of reactive oxygen species. A differential alkylation of cysteine residues in MT of the heart extracts was performed. Free and metal-bound cysteines were first trapped by N-ethylmaleimide and the disulfide bonds were reduced by dithiothreitol followed by alkylation with radiolabeled iodoacetamide. Analyses of the differentially alkylated MTs in the heart extract by high preformance liquid chromatography, SDS-PAGE, Western blot, and mass spectrometry revealed that disulfide bonds were present in MT in vivo under both physiological and oxidative stress conditions. More disulfide bonds were found in MT under the oxidative stress conditions. The MT disulfide bonds were likely intramolecular and both α- and β-domains were involved in the disulfide bond formation, although the α-domain appeared to be more easily oxidized than the β-domain. The results suggest that under physiological conditions, the formation of MT disulfide bonds is involved in the regulation of zinc homeostasis. Additional zinc release from MT under oxidative stress conditions is accompanied by more MT disulfide bond formation.

Original languageEnglish (US)
Pages (from-to)681-687
Number of pages7
JournalJournal of Biological Chemistry
Volume281
Issue number2
DOIs
StatePublished - Jan 13 2006
Externally publishedYes

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Oxidative stress
Metallothionein
Disulfides
Transgenic Mice
Oxidative Stress
Zinc
Alkylation
Cysteine
Iodoacetamide
Physiological Stress
Ethylmaleimide
Dithiothreitol
Liquid Chromatography
Doxorubicin
Liquid chromatography
Polyacrylamide Gel Electrophoresis
Cultured Cells
Reactive Oxygen Species
Mass Spectrometry
Homeostasis

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Metallothionein disulfides are present in metallothionein-overexpressing transgenic mouse heart and increase under conditions of oxidative stress. / Feng, Wenke; Benz, Frederick W.; Cai, Jian; Pierce, William M.; Kang, Yujian.

In: Journal of Biological Chemistry, Vol. 281, No. 2, 13.01.2006, p. 681-687.

Research output: Contribution to journalArticle

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