Regulation of microsomal and cytosolic glutathione S-transferase activities by S-nitrosylation

Abstract

There is increasing evidence that S-nitrosylation is a mechanism for the regulation of protein function via the modification of critical sulfhydryl groups. The activity of rat liver microsomal glutathione S-transferase (GST) is increased after treatment with N-ethylmaleimide (NEM), a sulfhydryl alkylating reagent, and is also increased under conditions of oxidative stress. In the present study, preincubation of purified rat liver microsomal GST with S-nitrosoglutathione (GSNO) or the nitric oxide (NO) donor, 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO), resulted in a 2-fold increase in enzyme activity. This increase in activity was reversed by dithiothreitol. The initial treatment of microsomal GST with either GSNO or DEA/NO was associated with an 85% loss of free sulfhydryl groups. After removal of the nitrosylating agents over a 6-hr period, approximately 50% of the enzyme was still nitrosylated, as determined by redox chemiluminescence. Furthermore, preincubation of either purified enzyme or hepatic microsomes with GSNO or DEA/NO prevented further enzyme activation by NEM, suggesting that NEM and the NO donors interact with a common population of sulfhydryl groups in the enzyme. In contrast, both NEM and NO donors partially inhibited the activity of cytosolic GST isoforms. The inhibitory activity of NEM and NO donors was much more evident when the GST pi isoform was used instead of a mixture of GST isoforms. These data suggest that there may be differential regulation of microsomal and cytosolic GST activities under conditions of nitrosative stress.

Introduction

The GSTs are involved in the biotransformation of numerous carcinogenic, mutagenic, toxic, and pharmacologically active compounds [1]. These enzymes are found largely in the cytosol as homodimers or heterodimers, and in the rat over twenty different GST subunits have been identified [2]. A membrane-bound GST that accounts for up to 3% of microsomal protein also has been identified [3], [4]. However, this GST isoform bears no obvious structural resemblance (amino acid sequence, molecular weight, or immunological properties) to the cytosolic GSTs. The microsomal GST exists as a trimer of identical 17.2 kDa subunits [5], [6], [7], [8]. Unlike the cytosolic GSTs, microsomal GST activity is increased by partial proteolysis [9] or by sulfhydryl reagents, such as NEM, that bind covalently to the sole cysteine residue (Cys49) in each polypeptide [10]. This modification results in a 10- to 15-fold increase in enzyme activity. In addition, the redox state of sulfhydryl groups in microsomal GST can also regulate enzyme activity.

NO is an important signaling molecule in both physiological and pathological processes. S-Nitrosothiols, such as GSNO, may function as storage forms of NO in vivo, and may participate in transnitrosation reactions. It is becoming increasingly evident that S-nitrosylation is a mechanism for modifying protein function through alterations in the function of sulfhydryl groups. S-Nitrosylation of a variety of proteins has been demonstrated, ranging from ion channels, enzymes, transcription factors, G-proteins, and kinases. Since modification of the sulfhydryl group in microsomal GST can regulate its activity, we hypothesized that this enzyme could be another protein target of NO. On the other hand, it has been reported that GSNO inhibits cytosolic GST activity by competing with GSH for binding at the active site of the enzyme, rather than by covalent modification of the enzyme [11]. In this work, we investigated the effect of NO donors on rat liver microsomal and cytosolic GST activity by using NEM to monitor the state of sulfhydryl groups in the enzyme.