S-Mercuration of rat sorbitol dehydrogenase by methylmercury causes its aggregation and the release of the zinc ion from the active site
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We previously developed a screening method to identify proteins that undergo aggregation through S-mercuration by methylmercury (MeHg) and found that rat arginase I is a target protein for MeHg (Kanda et al. in Arch Toxicol 82:803–808, 2008). In the present study, we characterized another S-mercurated protein from a rat hepatic preparation that has a subunit mass of 42 kDa, thereby facilitating its aggregation. Two-dimensional SDS–polyacrylamide gel electrophoresis and subsequent peptide mass fingerprinting using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry revealed that the 42 kDa protein was NAD-dependent sorbitol dehydrogenase (SDH). With recombinant rat SDH, we found that MeHg is covalently bound to SDH through Cys44, Cys119, Cys129 and Cys164, resulting in the inhibition of its catalytic activity, release of zinc ions and facilitates protein aggregation. Mutation analysis indicated that Cys44, which ligates the active site zinc atom, and Cys129 play a crucial role in the MeHg-mediated aggregation of SDH. Pretreatment with the cofactor NAD, but not NADP or FAD, markedly prevented aggregation of SDH. Such a protective effect of NAD on the aggregation of SDH caused by MeHg is discussed.
KeywordsMethylmercury Covalent modification Cysteine Aggregation Sorbitol dehydrogenase
Achromobacter protease I
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
Coomassie brilliant blue
We thank Dr. Daigo Sumi (Faculty of Pharmaceutical Sciences, Tokushima Bunri University) and Dr. Akira Yasutake (National Institute for Minamata Disease) for helpful advice; Dr. Nobuhiro Shimojo (University of Tsukuba) for his encouragement; Drs. Tomomi Gotoh and Masataka Mori (Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University) for donating the antibody against arginase I. This work was supported by a grant-in-aid (23117703 to Y.K. for scientific research) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Conflict of interest
The authors declare that they have no conflict of interest.
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