Abstract
Dermal 7alpha- and 7beta-hydroperoxycholest-5-en-3beta-ols (cholesterol 7alpha- and 7beta-hydroperoxides), regarded as good aging markers in the rat (Ozawa, N., Yamazaki, S., Chiba, K., Aoyama, H., Tomisawa, H., Tateishi, M., and Watabe, T. (1991) Biochem. Biophys. Res. Commun. 178, 242-247), were reduced in the presence of glutathione (GSH) with concomitant formation of GSSG by cytosol from rat liver in which no detectable level of the hydroperoxides had been demonstrated to occur. The GSH peroxidase (GSH Px) activity toward the toxic steroid hydroperoxides was exerted to almost the same extent by both Alpha-class GSH S-transferases (GSTs), Ya-Ya and Ya-Yc, and by selenium-containing GSH Px (Se-GSH Px) in rat liver cytosol. None of three Mu-class GSTs, Yb1-Yb1, Yb1-Yb2, and Yb2-Yb2, and a Theta-class GST, Yrs-Yrs, from rat liver and a Pi-class GST, Yp-Yp, from rat kidney showed any appreciable GSH Px activity toward the hydroperoxides. The subunit Ya-bearing GSTs and Se-GSH Px purified from rat liver cytosol showed marked differences in apparent specific activity toward the cholesterol hydroperoxides (GSTs Ya-Ya > Ya-Yc >> Se-GSH Px). However, a kinetic study indicated that Se-GSH Px had a higher affinity for steroid hydroperoxides than did the GSTs, so that Se-GSH Px could catalyze the reduction of lower concentrations of cholesterol 7-hydroperoxides with approximately equal Vmax/Km values to those by the GSTs. Rat skin had no GST bearing the subunit Ya but contained only a very low concentration of Se-GSH Px, possibly resulting in the accumulation of cholesterol 7-hydroperoxides in the skin but not in the liver. From rat skin cytosol, GSTs Yc-Yc, Yb1-Yb1, Yb1-Yb2, Yb2-Yb2, and Yp-Yp were isolated, purified to homogeneity, and identified with the corresponding GSTs from liver and kidney. The GSTs accounted for 0.23% of total skin cytosolic protein, and the most abundant isoform of skin GSTs was Yb2-Yb2, followed by Yc-Yc, Yp-Yp, Yb1-Yb1, and Yb1-Yb2 in decreasing order.
Highlights
Dermal 7␣- and 7-hydroperoxycholest-5-en-3-ols, regarded as good aging markers in the rat (Ozawa, N., Yamazaki, S., Chiba, K., Aoyama, H., Tomisawa, H., Tateishi, M., and Watabe, T. (1991) Biochem
The present study strongly suggests that the reason that the accumulation of cholesterol 7-hydroperoxides in skin is a good aging marker in rats may be in large part attributable to the absence of the Alpha-class GSH S-transferases (GSTs) bearing subunit Ya (Fig. 4) and to the presence of only a very low concentration of Se-GSH peroxidase (GSH Px) in dermal tissue (Table IV)
Ya-Yc could catalyze the reduction of cholesterol 7-hydroperoxides (Table I). These Alpha-class GSTs have been demonstrated to be isoforms accounting for 9.6 and 10% of total GSTs for Ya-Ya and Ya-Yc, respectively, in rat liver which contains no detectable level of the steroid hydroperoxides [34]
Summary
Materials—1-Chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), 30% (w/w) hydrogen peroxide, yeast GSH reductase, and NADPH were purchased from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan) and cholesterol, ethacrynic acid, phenylmethylsulfonyl fluoride, cumene hydroperoxide, linoleic acid, linolenic acid, arachidonic acid, S-hexyl-GSH, and Mr marker proteins were from Sigma, DE52 from Whatman Ltd. (Maidstone, United Kingdom), and CM-Sephadex C-50, DEAE Sephadex A-50, Sephadex G-200, epoxy-activated Sepharose 6B, activated thiol-Sepharose 4B, PBE 94, PBE 118, blue Sepharose 6B, Pharmalyte 8 –10.5, and Polybuffer 96 from Pharmacia Biotech Inc. (Uppsala, Sweden). The chromatographic fractions active toward hydrogen peroxide were pooled and concentrated to 7 ml (542 mg of protein) against Aquacide II and dialyzed against 5,000 ml of 20 mM sodium phosphate buffer, pH 6.0, containing 0.7 mM 2-mercaptoethanol and 1 mM EDTA (buffer B) for 18 h, and applied to a CM-Sephadex C-50 column (1.7 ϫ 21 cm) equilibrated with buffer B. The chromatographic fractions active toward hydrogen peroxide were pooled and concentrated to 5 ml (108 mg of protein) against Aquacide II and dialyzed against 5,000 ml of 10 mM sodium phosphate buffer, pH 7.0, containing 1 mM EDTA (buffer C) for 18 h and applied to an activated thiol-Sepharose 4B column (0.9 ϫ 5 cm) which was pre-equilibrated with buffer C, containing 0.1 M NaCl. The enzyme adsorbed was eluted from the column with 0.3 mM cysteine in buffer C after the column was washed with buffer C (30 ml) containing 0.1 M NaCl. The column effluent was collected into 2-ml fractions. Protein Determination—Protein concentration was determined by the method of Lowry et al [32] with bovine serum albumin as a standard
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