In vitro kinetics of hepatic glutathione S-transferase conjugation in largemouth bass and brown bullheads

The kinetics of glutathione S-transferase (GST) catalysis were investigated in largemouth bass (Micropterus salmoides) and brown bullheads (Amerius ne bulosus), two freshwater fish species found in a variety of polluted waterw ays in the eastern United States. The initial rates of hepatic GST activity toward four GST substrates, including 1-chloro-2,4-dinitrobenzene, ethacry nic acid, Delta 5-androstene-17-dione, and nitrobutyl chloride, were signif icantly higher in brown bullheads than in largemouth bass. Hepatic GST acti vity toward 1,2-dichroro-4-nitrobenzene, a mu-class GST substrate in rodent s, was not detectable in either species. Liver cytosolic GSTs were more eff icient in bullheads than in bass at catalyzing 1-chloro-2,4-dinitrobenzene- reduced glutathione (CDNB-GSH) conjugation over a broad range of electrophi le (CDNB) concentrations, including those representative of environmental e xposure. In contrast, largemouth bass maintained higher ambient concentrati ons of GSH, the nucleophilic cofactor for GST-mediated conjugation, than br own bullheads. Biphasic kinetics for GST-CDNB conjugation under conditions of variable GSH concentration were apparent in Eadie-Hofstee plots of the k inetic data, suggesting the presence of at least two hepatic GST isozymes w ith markedly different K-m values for GSH in both species. The GST-CDNB rea ction rate data obtained under conditions of variable GSH were well fitted (R-2 = 0.999) by the two-enzyme Michaelis-Menten equation. In addition, Wes tern blotting experiments confirmed the presence of two different hepatic G ST-like proteins in both largemouth bass and brown bullhead liver. Collecti vely, these findings indicate that largemouth bass and brown bullhead GSTs catalyze the conjugation of structurally diverse, class-specific GST substr ates, and that brown bullheads exhibit higher initial rates of GST activity than largemouth bass. The relatively higher rates of in vitro liver GST ac tivity at the low substrate concentrations relevant to environmental exposu re is expected to protect brown bullheads from the toxic effects of sedimen t-associated electrophilic chemicals. The somewhat lower rates of GST activ ity in largemouth bass liver compared with brown bullhead liver however, ma y be offset by maintenance of higher ambient hepatic GSH concentrations in largemouth bass.