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J. Biol. Chem., Vol. 281, Issue 2, 681-687, January 13, 2006

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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, and Y. James Kang1

From the Departments of Medicine and Pharmacology and Toxicology, University of Louisville, Louisville, Kentucky 40292

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 - and -domains were involved in the disulfide bond formation, although the -domain appeared to be more easily oxidized than the -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.

Received for publication, June 27, 2005 , and in revised form, September 23, 2005.

^* This work was supported in part by National Institutes of Health Grants HL59225 and HL63760 (to Y. J. K.) and Kentucky Science and Engineering Foundation Grant KSEF-148-502-03-81 (to W. F. and Y. J. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Distinguished University Scholar of the University of Louisville. To whom correspondence should be addressed: 511 S. Floyd St., MDR530 Louisville, KY 40202. Tel.: 502-852-8677; Fax: 502-852-6904; E-mail: yjkang01{at}louisville.edu.

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