Glutathione S-transferases in elasmobranch liver. Molecular heterogeneity, catalytic and binding properties, and purification

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Abstract
In order to gain insight into the phylogeny and physiological significance of organic-anion-binding proteins in the liver, the hepatic glutathione S-transferases of rat and a typical elasmobranch, the thorny-back shark (P. triseriata), were compared with respect to both glutathione S-transferase activities and organic-anion-binding properties. On gel filtration (Sephadex G-75, Superfine grade) of rat cytosol, the elution volumes of enzyme activities with 1-chloro-2,4-dinitrobenzene and p-nitrobenzyl chloride as substrates were identical (rat Y-fractions; MW 45,000). In contrast, 2 peaks of enzyme activity for 1-chloro-2,4-dinitrobenzene with elution volumes corresponding to MW 52,000 (PLAT Y1) and MW 45,000 (PLAT Y2) were detected on gel filtration of P. triseriata cytosol. Only fraction PLAT Y2 had enzyme activity with p-ntitrobenzyl chloride. Enzyme kinetics studies showed that rat Y-fraction had higher affinities for both 1-chloro-2,4-dinitrobenzene and glutathione than PLAT Y1- and PLAT Y2-fractions. The 2 forms of P. triseriata glutathione S-transferases differed greatly in affinity for glutathione. At a glutathione concentration that was physiological in P. triseriata, PLAT Y2 accounted for .apprx. 70% of the total glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene. Binding studies revealed that PLAT Y1 and PLAT Y2 fractions had much lower affinities for sulfobromophthalein and bilirubin than rat Y-fraction. In contrast, binding affinities of PLAT Y1 and PLAT Y2 for Rose Bengal and 1-anilino-8-naphthalenesulfonate were comparable with that of rat Y-fraction. Inhibitory kinetics suggested that sulfobromophthalein and Rose Bengal were non-competitive inhibitors of glutathione S-transferase activities when 1-chloro-2,4-dinitrobenzene was used as substrate for both PLAT Y1 and PLAT Y2. The major glutathione S-transferase from the PLAT Y2 fraction was purified 81-fold by sequential chromatography on Sephadex G-75, DEAE-Sephadex and hydroxyapatite, and consisted of 2 identical subunits with pI [isoelectric point] 7.7. The highly enriched Y2-fraction retained high affinity binding of Rose Bengal and 1-anilino-8-naphthalenesulfonate.