Proteomic analysis of glutathione S-transferase isoforms in mouse liver mitochondria.
OBJECTIVE
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.


CONCLUSIONS
Our results indicate that GSTs exist widely in mitochondria and its abundances of mitochondrial GSTs might be tissue-dependent and disease-related.
Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 101318, China.
Hai-Dan
HD
Sun
Ya-Wei
YW
Ru
Dong-Juan
DJ
Zhang
Song-Yue
SY
Yin
Liang
L
Yin
Ying-Ying
YY
Xie
You-Fei
YF
Guan
Si-Qi
SQ
Liu
D000818:Animals; D002138:Calibration; D003921:Diabetes Mellitus, Experimental; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D005982:Glutathione Transferase / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008930:Mitochondria, Liver / Q000378:metabolism*; D020033:Protein Isoforms; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods
D020033:Protein Isoforms; D011994:Recombinant Proteins; D005982:Glutathione Transferase