Further evidence that rat liver microsomal glutathione transferase 1 is not a cellular protein target for S-nitrosylation.
By adopting biotin switch method, we recently reported that liver microsomal glutathione transferase 1 (MGST1) might not be a protein target for S-nitrosylation in rat microsomes or in vivo. However, alternative analytic methods are needed to confirm this observation, as a single biotin switch method in judging specific protein S-nitrosylation in biological samples is increasingly recognized as insufficient, or even unreliable. Besides, only MGST1 localized on endoplasmic reticulum (ER), but not mitochondria which favors protein S-nitrosylation was examined in the previous report. Present study was therefore carried out to address these issues. Primary cultured hepatocytes were used. A physiological existing nitric oxide (NO) donor S-nitrosoglutathione (GSNO) was adopted to trigger protein S-nitrosylation. MGST1 was immunoprecipitated and its S-nitrosothiol content was measured by the NO probe 2,3-diaminonaphthalene. In parallel, S-nitrosylated proteins were immunoprecipitated by a monoclonal anti-S-nitrosocysteine antibody and probed with an anti-MGST1 antibody. In hepatocytes, neither ER nor mitochondria were found to contain S-nitrosylated MGST1 after GSNO treatment, showing that differently distributed MGST1 was consistently un-nitrosylable in the cellular environment. But under broken cell conditions, when samples were incubated directly with GSNO, MGST1 S-nitrosylation was indeed detectable in both the microsomal and mitochondrial proteins, indicating that previous failure in detecting MGST1 S-nitrosylation in microsomes is due to the limitations of biotin switch method. These results clearly, if not definitely, demonstrate that MGST1 is not a ready candidate for S-nitrosylation in the cellular content, despite its susceptibility to S-nitrosylation under broken cell conditions.
Shi
Qiang
Q
Institute of Pharmacology & Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310031, China.
Chen
Hai-Fei
HF
Lou
Yi-Jia
YJ
D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D005982:Glutathione Transferase / Q000378:metabolism*; D022781:Hepatocytes / Q000201:enzymology; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D009589:Nitrogen Oxides / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D026422:S-Nitrosoglutathione / Q000378:metabolism
D009589:Nitrogen Oxides; C026373:2,3-diaminonaphthalene; D026422:S-Nitrosoglutathione; D015081:2-Naphthylamine; D005982:Glutathione Transferase