Heterogeneity of dog-liver glutathione S-transferases. Evidence for a unique temperature dependence of the catalytic process.
Dog liver glutathione S-transferase activities are associated with five cytosolic proteins and to approximately 1.5% with microsomal proteins determined on the basis of activity conjugating to 1-chloro-2,4-dinitrobenzene. The four major cytosolic enzymes were purified to apparent homogeneity by sequential use of ion-exchange, hydrophobic, hydroxyapatite and affinity chromatography. The isolated transferases are binary combinations of three classes of subunits: alpha (Mr = 26,000), beta (Mr = 27,000), gamma (Mr = 28,500). They were classified by roman numerals assigned in order of increasing isoelectric point as DI alpha gamma (pI 6.4), DII alpha alpha (pI 6.9), DIII beta gamma (pI 8.1), and DIV beta gamma (pI 8.7). Additionally, traces of conjugating activity may be attributed to a, beta monomeric or dimeric protein with cationic character. The differences in catalytic specificity, temperature and pH dependence of activity, and sensitivity and kinetic response to inhibitory ligands may reflect the intrinsic structural heterogeneity of the transferases. At physiological glutathione concentrations DI alpha gamma accounted for roughly 60% of the total 1-chloro-2,4-dinitrobenzene-conjugating activity, the rank order of activity being DI alpha gamma greater than DII alpha alpha greater than DIV beta gamma greater than DIII beta gamma. The glutathione-dependent denitration of organic nitrates seems to be restricted to the cationic enzymes, whereas 1,2-dichloro-4-nitrobenzene-conjugating activity is exclusively associated with the anionic transferases, DI alpha gamma much greater than DII alpha alpha. Arrhenius plots from initial rate experiments performed over a range of temperatures (15-40 degrees C) exhibit an upward bend for DI alpha gamma, an apparently constant slope for DII alpha alpha and DIII beta gamma, and a downward bend for DIV beta gamma.
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D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D000818:Animals; D001663:Bilirubin / Q000494:pharmacology; D001961:Bromcresol Green / Q000494:pharmacology; D002850:Chromatography, Gel; D003840:Deoxycholic Acid / Q000494:pharmacology; D004285:Dogs; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D008970:Molecular Weight; D011339:Probenecid / Q000494:pharmacology; D013379:Substrate Specificity; D013448:Sulfobromophthalein / Q000494:pharmacology; D013696:Temperature
D000817:Anilino Naphthalenesulfonates; D007527:Isoenzymes; D046911:Macromolecular Substances; D003840:Deoxycholic Acid; D013448:Sulfobromophthalein; C027132:1-anilino-8-naphthalenesulfonate; D001961:Bromcresol Green; D005982:Glutathione Transferase; D011339:Probenecid; D001663:Bilirubin