In vitro kinetics of hepatic glutathione s‐transferase conjugation in largemouth bass and brown bullheads

Abstract

The kinetics of glutathione S‐transferase (GST) catalysis were investigated in largemouth bass (Micropterus salmoides) and brown bullheads (Amerius nebulosus), two freshwater fish species found in a variety of polluted waterways in the eastern United States. The initial rates of hepatic GST activity toward four GST substrates, including 1‐chloro‐2,4‐dinitrobenzene, ethacrynic acid, Δ5‐androstene‐17‐dione, and nitrobutyl chloride, were significantly higher in brown bullheads than in largemouth bass. Hepatic GST activity toward 1,2‐dichloro‐4‐nitrobenzene, a μ‐class GST substrate in rodents, was not detectable in either species. Liver cytosolic GSTs were more efficient in bullheads than in bass at catalyzing 1‐chloro‐2,4‐dinitrobenzene‐reduced glutathione (CDNB‐GSH) conjugation over a broad range of electrophile (CDNB) concentrations, including those representative of environmental exposure. In contrast, largemouth bass maintained higher ambient concentrations of GSH, the nucleophilic cofactor for GST‐mediated conjugation, than brown bullheads. Biphasic kinetics for GST‐CDNB conjugation under conditions of variable GSH concentration were apparent in Eadie‐Hofstee plots of the kinetic data, suggesting the presence of at least two hepatic GST isozymes with markedly different Km values for GSH in both species. The GST‐CDNB reaction rate data obtained under conditions of variable GSH were well fitted (R2 = 0.999) by the two‐enzyme Michaelis‐Menten equation. In addition, Western blotting experiments confirmed the presence of two different hepatic GST‐like proteins in both largemouth bass and brown bullhead liver. Collectively, these findings indicate that largemouth bass and brown bullhead GSTs catalyze the conjugation of structurally diverse, class‐specific GST substrates, and that brown bullheads exhibit higher initial rates of GST activity than largemouth bass. The relatively higher rates of in vitro liver GST activity at the low substrate concentrations relevant to environmental exposure is expected to protect brown bullheads from the toxic effects of sediment‐associated electrophilic chemicals. The somewhat lower rates of GST activity in largemouth bass liver compared with brown bullhead liver, however, may be offset by maintenance of higher ambient hepatic GSH concentrations in largemouth bass.

Cloning, expression and analysis of the olfactory glutathione S-transferases in coho salmon

“…In order to establish the cross-reactivity and immunochemical identity of OlfGSTs, Western blotting was performed using polyclonal GST antisera from striped bass [24, 25] and channel catfish [26]. The striped bass GST antisera recognized OlfGST rho class proteins but not OlfGST pi or omega (Figure 3B), whereas the catfish GST antisera recognized OlfGST pi class proteins but not OlfGST rho and omega (Figure 3C).…”

“…Proteins were either visualized by staining with Bio-Safe ™ Coomassie G-250 Stain (Bio-Rad, Hercules, CA) or transferred to a Hybond ™ -P PVDF membrane (GE Healthcare-Biosciences, Piscataway, NJ) prior to analysis by Western blotting. Membranes were either incubated with a 1:5,000 dilution of polyclonal striped bass GST antisera [24, 25] or a 1:5,000 dilution of polyclonal channel catfish GST antisera [26] in 1X PBS-0.1% Tween containing 5% non-fat dried milk (Bio-Rad, Hercules, CA). Binding of the immobilized proteins was detected with 1:10,000 dilution of donkey anti-rabbit IgG antibody conjugated with horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA) and enhanced chemiluminescence (GE Healthcare-Biosciences, Piscataway, NJ).…”

“…The initial rate enzymatic activities of coho hepatic cytosolic GST toward the reference substrates CDNB, ECA, CuOOH, and DCNB were determined using a SpectraMax190 UV–vis 96-well microplate reader after determining pH optima for each substrate [9, 25]. All catalytic activity assays were performed at 30 °C and corrected for non-enzymatic activity.…”

“…(B–C). Samples were blotted on PVDF membrane and probed with polyclonal GST antisera raised against striped bass GST that recognizes multiple GST forms, including rho of bass [24, 25] and also against a channel catfish GST pi antibody [26]. In both (B) and (C), lane 2 contained coho salmon olfactory cytosol (10 μg); and lanes 3–5 contained 2.5 μg of recombinant OlfGST pi, rho, and omega respectively.…”

In vitro hepatic metabolism of 2,2′,4,4′,5-pentabromodiphenyl ether (BDE 99) in Chinook Salmon (Onchorhynchus tshawytscha)

“…Mean NADPH-cytochrome C reductase activity for Chinook salmon cytosolic fractions was 3 ±0.5 nmol, min −1 , mg protein −1 , and for carp cytosolic fractions was 6 ±0.9 nmol, min −1 , mg protein −1 . Cytosolic glutathione S -transferase activities were determined utilizing the broad GST substrate CDNB (Habig and Jakoby, 1981) modified for a microplate reader (Gallagher et al, 2000). Mean GST-CDNB activity for Chinook cytosolic fractions was 303 ±74 nmol, min −1 , mg protein −1 and was 310 ±104 nmol, min − , mg protein −1 for carp cytosolic fractions.…”