Glutathione S-transferases in the adrenal cortex

• Main Author: Meikle, Ian
• Published: University of Edinburgh 1992
• Subjects: 571.1
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.657674

Data available prior to this thesis had shown that, of all bovine organs examined, the adrenal cortex exhibited the second highest level of glutathione S-transferase (GST) expression behind the liver. This finding, along with increasing evidence implicating the importance of GST in endogenous detoxification processes, formed the basis for a further extensive investigation of the GST isoenzymes expressed by the adrenal cortex. Investigation of the GST isoenzymes expressed by a number of different bovine organs using affinity chromatography on S-hexylglutathione-Sepharose 6B (S-hexG-Ag) revealed a marked organ-specific distribution of these enzymes. Bovine adrenal cortex, in particular, expressed isoenzymes from each GST class, as determined by immunoblotting experiments. GST activity determinations of these enzyme pools using a number of model substrates revealed the bovine enzymes to possess a specificity distinct to that of rat and human GST. Isoelectric focusing of the bovine adrenal cortex isoenzymes showed them to possess pl values similar to those found in other species. The affinity-purified mu- and pi-glass isoenzymes were resolved using anion-exchange chromatography, followed by reverse-phase hplc. Using this approach, at least 3 mu-class GST subunits and 1 pi-class GST subunit were identified. Ion-exchange chromatography failed to resolve the affinity-purified alpha-class GSTs, and reverse-phase hplc analysis resolved 2 polypeptides, designated Ya<SUB>1</SUB> and Ya<SUB>3</SUB> respectively. Analysis of the protein that failed to bind to the S-hexG-Ag column revealed that about 35% of GST activity remained in this fraction. Application of this material to glutathione-Sepharose 6B (GSH-Ag) resulted in the purification of an abundant alpha-class GST (1.3% total cytosolic protein). This GST was found to exhibit marked peroxidase and Δ5-ketosteroid isomerase activities, in addition to high activity with 4-hydroxynonenal. SDS/PAGE analysis revealed 2 distinct polypeptides of Mr 25900 and 26500, the former being equivalent to the Ya<SUB>3</SUB> subunit purified on S-hexG-Ag, and the latter named Ya<SUB>2</SUB>. Ion-exchange chromatography of the GSH-Ag purified alpha-class GST isoenzyme pool resulted in a complex picture, suggesting there to be at least 3 distinct subunits in this pool.