Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 101318, China.
World J Gastroenterol. 2012 Jul 14;18(26):3435-42. doi: 10.3748/wjg.v18.i26.3435.
To survey glutathione (GSH) S-transferase (GST) isoforms in mitochondria and to reveal the isoforms' biological significance in diabetic mice.
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.
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.
Our results indicate that GSTs exist widely in mitochondria and its abundances of mitochondrial GSTs might be tissue-dependent and disease-related.
研究谷胱甘肽 S-转移酶（GST）同工酶在鼠肝线粒体中的分布，并揭示其在糖尿病鼠中的生物学意义。
采用两种蛋白质组学方法，即谷胱甘肽亲和层析/二维电泳（2DE/MALDI-TOF/TOF-MS）和 SDS-PAGE/LC ESI-MS/MS，系统筛选鼠肝线粒体中 GST 的存在。使用针对 GST 的单克隆抗体进行 Western 印迹进一步证实蛋白质组学结果。为了定量评估肝线粒体 GSTs，通过各个重组 GST 蛋白的加载量与 Western 印迹中产生的相对强度生成校准曲线。在正常和 db/db 糖尿病鼠之间进行了广泛的肝线粒体 GST 比较。采用 Student's t 检验估计回归和显著性差异。
使用 GSH 亲和/2DE/MALDI-TOF/TOF-MS，鉴定出 3 种 GST，即 alpha3、mu1 和 pi1；而使用 SDS-PAGE/LC ESI-MS/MS 在鼠肝线粒体中检测到 5 种 GST，即 alpha3、mu1、pi1、kappa1 和 zeta1，其中 GST kappa1 被报道为一种特异的线粒体 GST。回归的 R²值介于约 0.86 和 0.98 之间，这对于定量是可以接受的。基于正常和糖尿病鼠肝线粒体中 GST 丰度的测量，发现 alpha3、kappa1、mu1 和 zeta1 这 4 种 GST 在两组动物之间几乎相当，而在糖尿病鼠中 GST pi1 的含量较低，与正常组相比，Western 印迹在对照和 db/db 糖尿病鼠肝线粒体中的信号分别为 134.61±53.84 与 99.74±46.2，P<0.05。
我们的结果表明 GST 广泛存在于线粒体中，其线粒体 GST 的丰度可能与组织和疾病有关。
