Abstract
The kinetics of the conjugation of glutathione (GSH) with anti-7beta, 8alpha-dihydroxy-9alpha,10alpha-oxy-7,8,9, 10-tetrahydrobenzo(a)pyrene (anti-BPDE) catalyzed by GSH S-transferase (GST) isoenzymes purified from the liver and forestomach of female A/J mouse has been investigated. The GST isoenzymes studied included an alpha class isoenzyme of forestomach (GST 9.5), alpha class hepatic isoenzymes mGSTA3-3 and mGSTA4-4, pi class hepatic isoenzyme mGSTP1-1, and mu class hepatic isoenzyme mGSTM1-1. When the concentration of (+)-anti-BPDE was varied (5-120 microM) at a fixed GSH concentration (2 mM), linear Lineweaver-Burk plots were observed for each isoenzyme. The kcat values for GST 9.5, mGSTA3-3, mGSTP1-1, mGSTM1-1, and mGSTA4-4 were 2.0, 0.02, 0.40, 0. 05, and 0.01 s-1, respectively, with corresponding Km values of 16, 12, 29, 27, and 49 microM. The catalytic efficiency (kcat/Km) of GST 9.5 in the conjugation of GSH with (+)-anti-BPDE, which is believed to be the ultimate carcinogenic metabolite of benzo(a)pyrene, was about 9-625-fold higher as compared with other mouse GST isoenzymes. These results indicate that GST 9.5 of forestomach is different among mammalian alpha class GSTs because (+)-anti-BPDE has been shown to be a poor substrate for alpha class rat or human GST isoenzymes. The catalytic efficiency of GST 9.5 was approximately 4.5-fold higher than that of pi class human isoenzyme (hGSTP1-1), which among human GSTs is reported to be most efficient in the detoxification of (+)-anti-BPDE. Unlike rat GST isoenzymes, linear Lineweaver-Burk plots were observed for mouse GSTs when GSH was used as a variable substrate. The catalytic efficiencies of the mouse GSTs toward (+)-anti-BPDE were about 2-20-fold higher as compared with the (-)-enantiomer of anti-BPDE. The results of the present study suggest that GST 9.5 may play an important role in the detoxification of (+)-anti-BPDE.
Highlights
Spread environmental pollutants, known as polycyclic aromatic hydrocarbons (PAH), which are thought to be etiological factors in human chemical carcinogenesis [1, 2]
Since BPDE is a poor substrate for epoxide hydrolase [12, 13, 18], the most important mechanism of BPDE inactivation seems to be its conjugation with GSH, a reaction catalyzed by glutathione S-transferases (GSTs) (EC 2.5.1.18) (14 –16)
Previous studies from our laboratory have shown that the GSH affinity purified GST preparations from the liver of female A/J mouse can be resolved into seven isoenzymes, which arise from different homo- or heterodimeric combinations of at least seven structurally distinct subunits
Summary
Materials—Female A/J mice (8 weeks old) were purchased from Jackson Laboratories (Bar Harbor, ME). Purification of GST Isoenzymes—GST isoenzymes of the liver and forestomach of female A/J mouse were purified by a protocol involving GSH-linked to epoxy-activated Sepharose 6B affinity chromatography followed by chromatofocusing. The homogeneity and classification of GST isoenzymes used for kinetic studies were ascertained by reverse-phase HPLC and Western blot analysis, respectively, as described by us previously.. Determination of GST Activity toward (ϩ)- and (Ϫ)-Anti-BPDE—A reverse-phase HPLC method was employed to study GST-catalyzed conjugation of GSH with anti-BPDE. Known amounts of (ϩ)- and (Ϫ)-anti-BPDE-[3H]GSH conjugates (3.3–203 pmol and 9.9 –119 pmol, respectively) were subjected to reverse-phase HPLC and monitored at 247 nm. GST-catalyzed conjugation of GSH with anti-BPDE was determined as a function of varying enzyme protein concentration for each isoenzyme to optimize incubation conditions. The GSH conjugates of anti-BPDE in the aqueous phase were quantitated by reverse-phase HPLC. A control without the enzyme protein was included to account for nonenzymatic conjugation of GSH with anti-BPDE
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