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Glutathione S-transferases in rat olfactory epithelium: purification, molecular properties and odorant biotransformation.

ABSTRACT: The olfactory epithelium is exposed to a variety of xenobiotic chemicals, including odorants and airborne toxic compounds. Recently, two novel, highly abundant, olfactory-specific biotransformation enzymes have been identified: cytochrome P-450olf1 and olfactory UDP-glucuronosyltransferase (UGT(olf)). The latter is a phase II biotransformation enzyme which catalyses the glucuronidation of alcohols, thiols, amines and carboxylic acids. Such covalent modification, which markedly affects lipid solubility and agonist potency, may be particularly important in the rapid termination of odorant signals. We report here the identification and characterization of a second olfactory phase II biotransformation enzyme, a glutathione S-transferase (GST). The olfactory epithelial cytosol shows the highest GST activity among the extrahepatic tissues examined. Significantly, olfactory epithelium had an activity 4-7 times higher than in other airway tissues, suggesting a role for this enzyme in chemoreception. The olfactory GST has been affinity-purified to homogeneity, and shown by h.p.l.c. and N-terminal amino acid sequencing to constitute mainly the Yb1 and Yb2 subunits, different from most other tissues that have mixtures of more enzyme classes. The identity of the olfactory enzymes was confirmed by PCR cloning and restriction enzyme analysis. Most importantly, the olfactory GSTs were found to catalyse glutathione conjugation of several odorant classes, including many unsaturated aldehydes and ketones, as well as epoxides. Together with UGT(olf), olfactory GST provides the necessary broad coverage of covalent modification capacity, which may be crucial for the acuity of the olfactory process.

SUBMITTER: Ben-Arie N

PROVIDER: S-EPMC1134220 | biostudies-other | 1993 Jun

REPOSITORIES: biostudies-other

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Project description: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 (Platyrhinoides triseriata), were compared with respect to both glutathione S-transferase activites 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; M(r) 45000). In contrast, two peaks of enzyme activity for 1-chloro-2,4-dinitrobenzene with elution volumes corresponding to M(r) 52000 (PLAT Y(1)) and M(r) 45000 (PLAT Y(2)) were detected on gel filtration of P. triseriata cytosol. Only fraction PLAT Y(2) had enzyme activity with p-nitrobenzyl chloride. Enzyme kinetic studies showed that rat Y-fraction had higher affinities for both 1-chloro-2,4-dinitrobenzene and glutathione than PLAT Y(1)- and PLAT Y(2)-fractions. The two forms of P. triseriata glutathione S-transferases differed greatly in affinity for glutathione. At a glutathione concentration that we found to be physiological in P. triseriata, PLAT Y(2) accounted for approx. 70% of the total glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene. Binding studies revealed that PLAT Y(1) and PLAT Y(2) fractions had much lower affinities for sulphobromophthalein and bilirubin than rat Y-fraction. In contrast, binding affinities of PLAT Y(1) and PLAT Y(2) for Rose Bengal and 1-anilino-8-naphthalenesulphonate were comparable with that of rat Y-fraction. Inhibitory kinetics suggested that sulphobromophthalein 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 Y(1) and PLAT Y(2). The major glutathione S-transferase from the PLAT Y(2) fraction was purified 81-fold by sequential chromatography on Sephadex G-75, DEAE-Sephadex and hydroxyapatite, and consisted of two identical subunits with pI7.7. The highly enriched Y(2)-fraction retained high affinity binding of Rose Bengal and 1-anilino-8-naphthalenesulphonate.

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Glutathione S-transferases in rat olfactory epithelium: purification, molecular properties and odorant biotransformation.

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