Purification and Characterization of Class μ Glutathione S-Transferase Isozymes from Rabbit Hepatic Tissue

Abstract

Class μ glutathione S-transferases (GSTs) are important in the detoxication of epoxides generated by oxidative metabolism. Phenobarbital, 3-methylcholanthrene, and pyridine have failed to enhance the expression of class μ GST isozymes in rabbit hepatic tissue (T. Primiano, S. G. Kim, and R. F. Novak, Toxicol. Appi. Pharmacol., 113, 64-73, 1992). Two class μ GST isozymes have been isolated from rabbit hepatic cytosol and purified to homogeneity using S-hexylglutathioneagarose, CM-Sepharose, and PBE94 chromatofocusing chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses showed that both isozymes possessed M r values of ≍25,500 and cross-reacted with class μ-specific GST IgG. Gel filtration analysis revealed that these isozymes were dimers with molecular weights of approximately 45 kDa. The class μ GST isozymes had pls of 7.8 and 7.2 as determined by nonequilibrium pH gel electrophoresis. The class μ GST 7.8 and 7.2 isozymes exhibited different metabolic activities toward the substrates 1-chloro-2,4-dinitrobenzene, bromosulfophthalein, 1,2-epoxy-3-( p-nitrophenoxy)propane, trans-4-phenyl-3-buten-2-one, p-nitrobenzyl chloride, and 3,4-dichloronitrobenzene. Metabolic activity of the two GSTs toward the substrate 1-chloro-2,4-dinitrobenzene was inhibited by Cibacron blue, triethyltin bromide, S-hexylglutathione, bromosulfophthalein, and indomethacin. The amino acid composition of GST μ 7.8 and 7.2 was determined and found to be very similar to those of purified rat class μ GST isozymes. N-terminal analysis of the first 21 residues of the p I 7.8 class μ GST isozyme revealed that it had 71 and 81% sequence identity with the Yb1 and Yb2 subunits, respectively. Similarly, N-terminal analysis of the first 21 residues of the p I 7.2 class μ GST isozyme revealed a 75% sequence identity with either the rat Yb1 or Yb2 subunit. Examination of class μ GST expression in rabbit hepatic cytosol following treatment with a series of known inducers including phenobarbital, 3-methyl-cholanthrene, isosafrole, pyrazine, trans-stilbene oxide, butylated hydroxyanisole, and tert-butyihydroquinone was accomplished. The data show that these agents not only failed to enhance class μ GST expression, but that 3-methylcholanthrene and isosafrole caused suppression of class μ GSTs. These results provide evidence for the existence of two closely related class μ GST isozymes in rabbit hepatic tissue and suggest that the molecular mechanisms regulating GST expression differ between rat and rabbit in response to these xenobiotics.