The unique feature of dog liver cytosolic glutathione S-transferases. An isozyme not retained on the affinity column has the highest activity toward 1,2-dichloro-4-nitrobenzene.
In the adult dog liver cytosol we identified four glutathione S-transferase (GST) subunits, Yd1 (Mr 26,000), Yd2 (Mr 27,000), Yd3 (Mr 28,000), and Ydf (Mr 27,400), and purified GST forms comprising Yd1, Yd2, and Yd3, to apparent homogeneity. Unlike rat transferases the enzyme activity toward 1,2-dichloro-4-nitrobenzene (DCNB) was not retained on the affinity column. Thus the DCNB-active enzyme, GST YdfYdf, from the flow-through fraction of the affinity column was also purified to homogeneity by gel filtration, DE52 chromatography, chromatofocusing, and hydroxylapatite column chromatography. Immunoblot analysis of dog GSTs revealed that the subunits Yd1, Yd2, and Yd3 belong to the pi, alpha, and mu class, respectively. On the contrary, Ydf had no reactivity with antibodies raised against any of the three classes of GST. Each subunit, Yd1, Yd2, Yd3, and Ydf, was distinguishable by its own retention time on reverse-phase high performance liquid chromatography. N-terminal amino acid sequences of the dog GSTS Yd1Yd1 and Yd3Yd3 revealed a high degree of homology to the pi and mu class transferases from rat, human, and mouse, respectively, while the N terminus of Yd2Yd2 is blocked. N-terminal amino acid sequences of GST YdfYdf showed no homology to any of the three classes of GST. The most significant property noted of GST YdfYdf is the high specific activity toward DCNB, exceeding by 1 order of magnitude the corresponding values for the known mu class GSTs. The present results strongly suggest that dog GST YdfYdf is a unique enzyme distinct from the hitherto characterized GST isozymes.
Laboratory of Biochemical Pharmacology and Biotoxicology, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
T
T
Igarashi
A
A
Kohara
Y
Y
Shikata
F
F
Sagami
J
J
Sonoda
T
T
Horie
T
T
Satoh
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D005779:Immunodiffusion; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009578:Nitrobenzenes / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity
D007527:Isoenzymes; D046911:Macromolecular Substances; D009578:Nitrobenzenes; C028328:1,2-dichloro-4-nitrobenzene; D005982:Glutathione Transferase