Glutathione S-transferases in the Japanese quail: tissue distribution and purification of the liver isozymes.
Cytosolic glutathione S-transferase (GST) activities toward 1-chloro-2,4-dinitro-benzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (EA), 1,2-epoxy-3-(p-nitrophenoxyl)propane (EPNP), trans-4-phenyl-3-buten-2-one(t-PBO), delta 5-androstene-3,17-dione (ASD) and trans-stilbene oxide (t-SO); cytosolic glutathione peroxidase activity toward cumene hydroperoxide (CuOOH); and microsomal GST activity toward CDNB were examined in liver, kidney, brain, and lung of adult male and female Japanese quail. In all cases, the renal specific activity per milligram protein was higher than the hepatic activity and was the highest among the four tissues examined. No consistent sex differences in GST activity were observed. The GSTs were purified from quail liver cytosol by S-hexylglutathione and glutathione affinity chromatography. Total GSTs eluted from the S-hexylglutathione affinity column were further separated by chromatofocusing, and the microheterogeneity of the GST isozymes was shown by high-resolution native isoelectrofocusing (IEF) in polyacrylamide slab gels and by SDS-PAGE. Five subunits were identified: QL1 (30.5 kDa), QL2 (27.2 kDa), QL3a (26.8 kDa), QL3b (26.5 kDa), and QL4 (25.5 kDa). Western blot analysis revealed that QL1 and QL2 reacted with antibodies raised against the rat Mu class GSTs (Yb1 and Yb2), and QL3a and QL3b reacted with those raised against the Alpha class (rat Ya and mouse a). Substrate specific activity of each isoform was determined with CDNB, DCNB, CuOOH, EA, t-PBO, ASD, and t-SO. QL3a and QL3b have high reactivity toward CuOOH, while QL1 and QL2 showed high activity toward t-SO. The N-terminal amino acid sequence of QL2 was identical to that of the chicken Mu class GST subunit CL2. However, no sequence was obtained with QL1 due to possible N-terminal blockage.
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Department of Entomology, North Carolina State University, Raleigh 27695-7647, USA.
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Roe
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D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D002074:Butanones / Q000378:metabolism; D003370:Coturnix; D003600:Cytosol / Q000201:enzymology; D004137:Dinitrochlorobenzene / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004852:Epoxy Compounds / Q000378:metabolism; D004976:Ethacrynic Acid / Q000378:metabolism; D005043:Etiocholanolone / Q000031:analogs & derivatives / Q000378:metabolism; D005260:Female; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007525:Isoelectric Focusing; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology*; D008168:Lung / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D009578:Nitrobenzenes / Q000378:metabolism; D009596:Nitrophenols / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013267:Stilbenes / Q000378:metabolism; D014018:Tissue Distribution
D002074:Butanones; D004137:Dinitrochlorobenzene; D004852:Epoxy Compounds; D009578:Nitrobenzenes; D009596:Nitrophenols; D011480:Protease Inhibitors; D013267:Stilbenes; C006576:androstane-3,17-dione; C007606:1,2-epoxy-3-(p-nitrophenoxy)propane; D005043:Etiocholanolone; C028328:1,2-dichloro-4-nitrobenzene; C025383:benzylideneacetone; D005982:Glutathione Transferase; C025906:stilbene oxide; D004976:Ethacrynic Acid