Mass spectrometric analysis of rat liver cytosolic glutathione S-transferases: modifications are limited to N-terminal processing

Abstract

Cytosolic glutathione S-transferases (GSTs) from rat livers were purified using an S-hexylglutathione affinity column. The GST subunits were resolved by reverse-phase HPLC and their molecular masses were determined by electrospray mass spectrometry. The major hepatic GSTs detected were subunits 1, 1′, 2, 3 and 4, with molecular mass of 25,520, 25,473, 25,188, 25,782 and 25,571 Da respectively. Subunits 6, 7 and 10 are minor components, with molecular mass of 25,551, 23,308 and 25,211 Da respectively. Alternatively, the hepatic GSTs were purified using a glutathione affinity column. Subunits 1, 1′, 2, 8 and 10 were eluted from this column with GSSG, the oxidized form of glutathione. Subunit 8 has a molecular mass of 25,553 Da. The remaining proteins on the glutathione affinity column were removed with glutathione and S-hexylglutathione. Subunits 2, 3, 4 and 6 could be detected in the eluate. We could not detect any significant difference in molecular mass between GSTs isolated from male and female rat livers. Cytosolic GSTs were isolated from livers of buthionine sulphoximine-treated female rats for MS analysis. The molecular masses obtained were identical to those determined for the controls.

The extended catalysis of glutathione transferase

“…Instead the values were estimated by extrapolating from experiments performed at higher reagent concentrations or at higher pH values, assuming a simple bimolecular interaction. 0.5 mM GSTs, approximately with the same isoenzyme composition found in rat liver (Alpha class GST = 0.27 mM, Mu class GST = 0.23 mM [12]), they become 20-30 times faster (Table 1 and Fig. 1).…”

“…At a pH value and GSH concentration similar to those found in the liver cytosol (pH 7.1, GSH 10 mM) these reactions are quite slow, showing apparent t 1/2 values ranging from 3 min to about 4 h (Table 1 ). When the same reactions were carried out in the presence of 0.5 mM GSTs, approximately with the same isoenzyme composition found in rat liver (Alpha class GST = 0.27 mM, Mu class GST = 0.23 mM [12]), they become 20–30 times faster (Table 1 and Fig. 1 ).…”

Conformational stability of pGEX‐expressed Schistosoma japonicum glutathione S‐transferase: A detoxification enzyme and fusion‐protein affinity tag

“…The cytosolic GSTs do not undergo posttranslational modification (Yeh et al, 1995), making E. coli heterologous expression systems suitable for their overexpression. Cytosolic GSTs are highly soluble proteins and GST fusions can be used as a strategy for expressing large quantities of soluble protein.…”

Changes in Glutathione Content in Liver Diseases: An Update

“…Alpha and Mu classes of GST2 superfamily enzymes, able to protect the cell from toxic endogenous or xenobiotic compounds, are abundantly expressed in rat liver, where they represent 43% and 56%, respectively, of the entire pool of cytosolic GSTs [54]. In rat hepatocytes, a significant fraction of dinitrosyl-diglutathionyl-iron complex is found in subcellular components, mainly in the nucleus, apparently entirely bound to Alpha class GSTs [55].…”