Brief Article
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World J Gastroenterol. Jul 14, 2012; 18(26): 3435-3442
Published online Jul 14, 2012. doi: 10.3748/wjg.v18.i26.3435
Proteomic analysis of glutathione S-transferase isoforms in mouse liver mitochondria
Hai-Dan Sun, Ya-Wei Ru, Dong-Juan Zhang, Song-Yue Yin, Liang Yin, Ying-Ying Xie, You-Fei Guan, Si-Qi Liu
Hai-Dan Sun, Ya-Wei Ru, Song-Yue Yin, Liang Yin, Ying-Ying Xie, Si-Qi Liu, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 101318, China
Dong-Juan Zhang, You-Fei Guan, Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing 100191, China
Author contributions: Sun HD and Liu SQ designed the study; Sun HD, Ru YW and Yin SY performed the research; Yin L and Xie YY analyzed the data; Zhang DJ and Guan YF provided the diabetes and normal mice; Sun HD and Liu SQ wrote the paper.
Supported by The National Basic Research Program of China, No. 2010CB912703; the Development Program of China, No. 2006AA02A308; and the Nature Science Foundation of China, No. 30900508
Correspondence to: Si-Qi Liu, PhD, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 101318, China. siqiliu@genomics.org.cn
Telephone: +86-10-80485327 Fax: +86-10-80485324
Received: December 24, 2011
Revised: February 6, 2012
Accepted: February 16, 2012
Published online: July 14, 2012
Abstract

AIM: To survey glutathione (GSH) S-transferase (GST) isoforms in mitochondria and to reveal the isoforms’ biological significance in diabetic mice.

METHODS: The presence of GSTs in mouse liver mitochondria was systematically screened by two proteomic approaches, namely, GSH affinity chromatography/two dimensional electrophoresis (2DE/MALDI TOF/TOF MS) and SDS-PAGE/LC ESI MS/MS. The proteomic results were further confirmed by Western blotting using monoclonal antibodies against GSTs. To evaluate the liver mitochondrial GSTs quantitatively, calibration curves were generated by the loading amounts of individual recombinant GST protein vs the relative intensities elicited from the Western blotting. An extensive comparison of the liver mitochondrial GSTs was conducted between normal and db/db diabetic mice. Student’s t test was adopted for the estimation of regression and significant difference.

RESULTS: Using GSH affinity/2DE/MALDI TOF/TOF MS, three GSTs, namely, alpha3, mu1 and pi1, were identified; whereas five GSTs, alpha3, mu1, pi1, kappa1 and zeta1, were detected in mouse liver mitochondria using SDS-PAGE/LC ESI MS/MS, of these GSTs, GST kappa1 was reported as a specific mitochondrial GST. The R2 values of regression ranged between values of about 0.86 and 0.98, which were acceptable for the quantification. Based on the measurement of the GST abundances in liver mitochondria of normal and diabetic mice, the four GSTs, alpha3, kappa1, mu1 and zeta1, were found to be almost comparable between the two sets of animals, whereas, lower GST pi1 was detected in the diabetic mice compared with normal ones, the signal of Western blotting in control and db/db diabetic mice liver mitochondria is 134.61 ± 53.84 vs 99.74 ± 46.2, with P < 0.05.

CONCLUSION: Our results indicate that GSTs exist widely in mitochondria and its abundances of mitochondrial GSTs might be tissue-dependent and disease-related.

Keywords: Glutathione S-transferase; Mitochondria; Liver; Proteomics; Diabetes