| Microsomal glutathione S-transferase 1 (MGST1) is one member of the membrane associated proteins in eicosanoid and glutathione metabolism super-family (MAPEG super-family) . Its functions involve catalyzing the conjugation between GSH and electrophiles, as well as the reduction of products from lipid-peroxidation, thus playing a protective role in the detoxification process and serving as a GSH-dependant peroxidase.The expression of MGST1 is relative stable both at the gene and protein level, as exemplified by the fact that traditional P450 inducers and inhibitors exert little effects on its expression. The most striking feature of MGST1 lies in that post-translational modifications on the enzyme could regulate its catalytic activities.MGST1 is a homo-trimeric protein, with each subunit possesses a molecular weight of 17.3 kDa and a single cyeteine residue at positive 49 ( Cys49) . Under physiological conditions, MGST1 displays very limited activities, however, a variety of post-translational modifications could significantly enhance its activities, causing enzyme activation. Early studies show that Cys49 is localized on the catalytic center of MGST1, and covalently modifications that occur on the sulfhydryl group in this residue could activate MGST1. Beside, both S-nitrosylation and oxidation, two otherforms of modifications on Cys49, could also lead to MGST1 activation.In addition to activation by Cys49, modifications on tyrosine residues also play an important role in the regulation of MGST1. MGST1 harbors seven tyrosines, but only Tyr92 is involved in the activation process. Modifications on tyrosine include nitration and crossing-linking forming a protein dimer.Reactive nitrogen species (RNS) refer to a variety of radicals containing nitrogen atom, and they can regulate many protein functions via post-translational modifications. It has been implicated that RNS, mainly peroxynitrite (ONOO~-) and S-nitrosoglutathione (GSNO), can also activate MGST1 by modifications on Cys49 or Tyr92.Modifications and activation of MGST1 are till now focused on in vitro experiments using purified enzyme or microsomal fractions. These studies might provide some insights into the in vivo importance of MGST1 activation, but significant hinder backs exist, i.e., the electron donor NADPH is ignored, the protein and cellular interactions are not considered. Those factors might change, or even regulate the in vivo modifications of MGST1. It is therefore of great interests to reveal the genuine mechanisms of MGST1 activations under in vivo conditions, so as to clarify the biological significance of MGST1 activation in the patho/physiological processes.The above-mentioned modifications of MGST1 are considered to be tissue-specific, i.e., activation occurs only in the livers of mammalian species, but not in the extra-hepatic tissues such as the intestine, the adrenal, and the testis. Therefore, present study adopted primary cultured rat hepatocytes as the experimental model for the investigations of MGST1 activation in a biological sample, attempting to clarify whether ONOO~- and GSNO could activate MGST1 under the cellular conditions.Part 1 Purification of rat liver MGSTl and preparation ofrabbitanti-MGSTl polyclonal antibodyAim: To establish the methods for both the purification of MGSTl and the generation of its antibody, thus providing the necessary material for subsequent experiments.Methods: (1) Rat liver microsomes were prepared by ultra-centrifuge. MGSTl was purified by chromatography on hydroxyapatite (HA) and CM-Sepharose CL-6B. SDS-PAGE was performed to examine the purity of MGSTl. (2) Purified MGSTl was blended with Freund's adjuvant;and the resulting water-in-oil emulsion was injected at the back of rabbit to trigger production of polyclonal antibody. Anti-MGSTl serum was test by dot blotting and Western blot.Results: (1) MGSTl was purified to a single protein band at 17 kDa on SDS-PAGE. (2 ) By using the conventional immunological methods, rabbit anti-MGSTl polyclonal antibody was successfully generated. This antibody can be adopted in the dot blotting and Western blot detection of MGSTl.Part 2 Activation of MGSTl by ONOO' in the primarycultured rat hepatocytes Aim: To observe whether MGSTl was dimerized in the liver total proteins or the primary cultured rat hepatocytes after ONOO" treatment. Whether similar modification occurs after hepatocytes damage was also examined. Methods: The liver total proteins or the primary cultured rat hepatocytes were incubated with ONOO" to trigger possible modifications on MGSTl. Western blot was carried out to observe the MGSTl dimers. Cellular fluorescence was measured to observe the changes of ONOO" after acetaminophen induced injury of hepatocytes;modification of MGSTl was monitored in this model as well.Results: Western blot showed that MGSTl formed a dimer after ONOO' treatment of either total liver proteins or the primary cultured hepatocytes. As for the model of acetaminophen overdose on hepatocytes, cellular ONOO" was dose-dependently increased, which is accompanied by the dimerization of MGSTl.Part 3 Modifications of MGSTl by GSNO on the primary cultured hepatocytes Aim: To observe whether MGSTl is S-nitrosylated after GSNO treatment of total liver proteins or primary cultured hepatocytes. S-nitrosylation of MGSTl in respond to the cellular injury by acetaminophen overdose is also explored. Methods: GSNO was incubated with either the total liver proteins or the primary cultured hepatocytes to trigger the S-nitrosylation of proteins. Immuno-precipitation of MGSTl from microsomal or mitochondria fractions was performed, followed by the DAN fluorescence detection to examined the S-nitrosylation of MGSTl. In parallel, the total S-nitrosylated proteins were immuno-precipitated from microsomal or mitochondria fractions, followed by SDS-PAGE and Western blot to detect the presence of MGSTl.Results: Both DAN fluorescence and Western blot showed that GSNO treatment of total liver proteins in vitro could trigger the S-nitrosylation of MGSTl, but treatment of primary cultured hepatocytes could not induce MGSTl S-nitrosylation, despite the fact the GSNO indeed caused an enhancement of S-nitrosylated proteins in both the microsomal and mitochondria fractions.Conclusions1. Successfully purified rat liver MGST1, and raised its polyclonal antibody.2. ONOO' could induce the dimerization of MGSTl in both the total liver proteins and the primary cultured rat hepatocytes.3. In the model of acetaminophen induced injury of primary cultured rat hepatocytes, MGSTl was dimerized, which might be mediated by ONOO" over-production.4. GSNO could trigger MGSTl S-nitrosylation in the total liver proteins, but not in the primary cultured rat hepatocytes.
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