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Activation and inhibition of microsomal glutathione transferase from mouse liver
C Andersson1, M Söderström, B Mannervik
1Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, Sweden.
Abstract:
Mouse liver microsomal glutathione transferase was purified in an N-ethylmaleimide-activated as well as an unactivated form. The enzyme had a molecular mass of 17 kDa and a pI of 8.8. It showed cross-reactivity with antibodies raised against rat liver microsomal glutathione transferase, but not with any of the available antisera raised against cytosolic glutathione transferases. The fully N-ethylmaleimide-activated enzyme could be further activated 1.5-fold by inclusion of 1 microM-bromosulphophthalein in the assay system. The latter effect was reversible, which was not the case for the N-ethylmaleimide activation. At 20 microM-bromosulphophthalein the activated microsomal glutathione transferase was strongly inhibited, while the unactivated form was activated 2.5-fold. Inhibitors of the microsomal glutathione transferase from mouse liver showed either about the same I50 values for the activated and the unactivated form of the enzyme, or significantly lower I50 values for the activated form compared with the unactivated form. The low I50 values and the steep slope of the activity-versus-inhibitor-concentration curves for the latter group of inhibitors tested on the activated enzyme indicate a co-operative effect involving conversion of activated enzyme into the unactivated form, as well as conventional inhibition of the enzyme.
Insights
Researchers purified mouse liver microsomal glutathione transferase in activated and unactivated forms. Bromosulphophthalein modulated enzyme activity, and inhibitors revealed cooperative effects in the activated form.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Microsomal glutathione transferase (MGST) plays a crucial role in cellular detoxification.
- Understanding the activation and inhibition mechanisms of MGST is vital for drug development and disease research.
Purpose of the Study:
- To purify and characterize mouse liver microsomal glutathione transferase in both activated and unactivated states.
- To investigate the effects of N-ethylmaleimide and bromosulphophthalein on enzyme activity.
- To analyze the inhibition kinetics of various compounds on different enzyme forms.
Main Methods:
- Purification of mouse liver microsomal glutathione transferase.
- Enzyme activity assays using N-ethylmaleimide and bromosulphophthalein.
- Western blot analysis using antibodies against rat liver MGST and cytosolic glutathione transferases.
- Inhibitor dose-response studies to determine I50 values.
Main Results:
- Mouse liver MGST was purified with a molecular mass of 17 kDa and a pI of 8.8.
- N-ethylmaleimide-activated enzyme showed further reversible activation by bromosulphophthalein, while the unactivated form was inhibited at higher concentrations.
- Inhibitor studies indicated cooperative effects and conversion between activated and unactivated enzyme forms.
Conclusions:
- Mouse liver MGST exhibits distinct properties in its activated and unactivated forms.
- Bromosulphophthalein differentially modulates MGST activity based on its activation state.
- Inhibitor interactions suggest complex regulatory mechanisms involving enzyme conformational changes.