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- Lindkvist, B, et al.
(author)
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Glutathione transferase mimics: micellar catalysis of an enzymic reaction
- 1997
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In: The Biochemical journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 323323 ( Pt 1), s. 39-43
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Journal article (peer-reviewed)abstract
- Substances that mimic the enzyme action of glutathione transferases (which serve in detoxification) are described. These micellar catalysts enhance the reaction rate between thiols and activated halogenated nitroarenes as well as α,β-unsaturated carbonyls. The nucleophilic aromatic substitution reaction is enhanced by the following surfactants in descending order: poly(dimethyldiallylammonium-co-dodecylmethyldiallylammon ium) bromide (86/14) ≫cetyltrimethylammonium bromide > zwittergent 3-16 (n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulphonate) > zwittergent 3-14 (n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulphonate) ≈ N,N-dimethyl-laurylamine N-oxide > N,N-dimethyloctylamine N-oxide. The most efficient catalyst studied is a polymeric material that incorporates surfactant properties (n-dodecylmethyldiallylammonium bromide) and opens up possibilities for engineering sequences of reactions on a polymeric support. Michael addition to α,β-unsaturated carbonyls is exemplified by a model substance, trans-4-phenylbut-3-en-2-one, and a toxic compound that is formed during oxidative stress, 4-hydroxy-2-undecenal. The latter compound is conjugated with the highest efficiency of those tested. Micellar catalysts can thus be viewed as simple models for the glutathione transferases highlighting the influence of a positive electrostatic field and a non-specific hydrophobic binding site, pertaining to two catalytic aspects, namely thiolate anion stabilization and solvent shielding.
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| 2. |
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| 3. |
- Sun, TH, et al.
(author)
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Binding of glutathione and an inhibitor to microsomal glutathione transferase
- 1997
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In: The Biochemical journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 326326 ( Pt 1), s. 193-196
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Journal article (peer-reviewed)abstract
- Microsomal glutathione transferase is an abundant liver protein that can be activated by thiol reagents. It is not known whether the activation is associated with changed binding properties of the enzyme. Therefore the binding of GSH and an inhibitor to rat liver microsomal glutathione transferase was studied by use of equilibrium dialysis and equilibrium partition in a two-phase system. The radioactive substrate glutathione and an inhibitor (glutathione sulphonate) give hyperbolic binding isotherms with a stoichiometry of 1 mol per mol of enzyme (i.e. 1 molecule per homotrimer). Glutathione had an equilibrium binding constant of 18 μM. Competition experiments involving glutathione sulphonate showed that it could effectively displace GSH. These and kinetic studies showed that the Kd and Ki for glutathione sulphonic acid are close to 10 μM. No change in these parameters was obtained after N-ethylmaleimide activation of the enzyme. Thus activation does not result from changes in binding affinity to GSH.
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| 4. |
- WEINANDER, R, et al.
(author)
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Heterologous expression of rat liver microsomal glutathione transferase in simian COS cells and Escherichia coli
- 1995
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In: The Biochemical journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 311311 ( Pt 3), s. 861-866
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Journal article (peer-reviewed)abstract
- The cDNA coding for rat liver microsomal glutathione transferase was subcloned into the mammalian expression vector pCMV-5 and the construct was transfected into, and transiently expressed in, simian COS cells. This resulted in high expression (0.7% of the microsomal protein). The activity towards 1-chloro-2,4-dinitrobenzene in microsomes was 15-30 nmol/min per mg, which increased upon N-ethylmaleimide treatment to 60-200 nmol/min per mg. Control and antisense-vector-treated cells displayed very low activity (3-6 nmol/min per mg). A DNA fragment coding for rat microsomal glutathione transferase was generated by PCR, cloned into the bacterial expression vector pSP19T7LT and transformed into Escherichia coli strain BL21 (DE3) (which contained the plasmid pLys SL). Isopropyl beta-D-thiogalactopyranoside (IPTG; 1 mM) induced the expression of significant amounts of enzymically active protein (4 mg/l of culture as measured by Western blots). The recombinant protein was purified and characterized and found to be indistinguishable from the rat liver enzyme with regard to enzymic activity, molecular mass and N-terminal amino acid sequence. Human liver cDNA was used to obtain the coding region of human microsomal glutathione transferase by PCR. This PCR product was cloned into pSP19T7LT, which, upon induction with IPTG, yielded significant amounts (9 mg/l of culture) of active enzyme in BL21 (DE3) cells. Thus, for the first time, it is now possible to express both human and rat microsomal glutathione transferase in an enzymically active form in Escherichia coli.
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