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- Bresell, Anders, et al.
(författare)
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Bioinformatic and enzymatic characterization of the MAPEG superfamily
- 2005
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 272:7, s. 1688-1703
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Tidskriftsartikel (refereegranskat)abstract
- The membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG) superfamily includes structurally related membrane proteins with diverse functions of widespread origin. A total of 136 proteins belonging to the MAPEG superfamily were found in database and genome screenings. The members were found in prokaryotes and eukaryotes, but not in any archaeal organism. Multiple sequence alignments and calculations of evolutionary trees revealed a clear subdivision of the eukaryotic MAPEG members, corresponding to the six families of microsomal glutathione transferases (MGST) 1, 2 and 3, leukotriene C4 synthase (LTC4), 5-lipoxygenase activating protein (FLAP), and prostaglandin E synthase. Prokaryotes contain at least two distinct potential ancestral subfamilies, of which one is unique, whereas the other most closely resembles enzymes that belong to the MGST2/FLAP/LTC4 synthase families. The insect members are most similar to MGST1/prostaglandin E synthase. With the new data available, we observe that fish enzymes are present in all six families, showing an early origin for MAPEG family differentiation. Thus, the evolutionary origins and relationships of the MAPEG superfamily can be defined, including distinct sequence patterns characteristic for each of the subfamilies. We have further investigated and functionally characterized representative gene products from Escherichia coli, Synechocystis sp., Arabidopsis thaliana and Drosophila melanogaster, and the fish liver enzyme, purified from pike (Esox lucius). Protein overexpression and enzyme activity analysis demonstrated that all proteins catalyzed the conjugation of 1-chloro-2,4-dinitrobenzene with reduced glutathione. The E. coli protein displayed glutathione transferase activity of 0.11 µmol·min−1·mg−1 in the membrane fraction from bacteria overexpressing the protein. Partial purification of the Synechocystis sp. protein yielded an enzyme of the expected molecular mass and an N-terminal amino acid sequence that was at least 50% pure, with a specific activity towards 1-chloro-2,4-dinitrobenzene of 11 µmol·min−1·mg−1. Yeast microsomes expressing the Arabidopsis enzyme showed an activity of 0.02 µmol·min−1·mg−1, whereas the Drosophila enzyme expressed in E. coli was highly active at 3.6 µmol·min−1·mg−1. The purified pike enzyme is the most active MGST described so far with a specific activity of 285 µmol·min−1·mg−1. Drosophila and pike enzymes also displayed glutathione peroxidase activity towards cumene hydroperoxide (0.4 and 2.2 µmol·min−1·mg−1, respectively). Glutathione transferase activity can thus be regarded as a common denominator for a majority of MAPEG members throughout the kingdoms of life whereas glutathione peroxidase activity occurs in representatives from the MGST1, 2 and 3 and PGES subfamilies.
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| 2. |
- Sun, Tie-Hua
(författare)
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Microsomal glutathione transferase : studies on the kinetic mechanism, species variety, binding properties and substrate measurement
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Microsomal glutathione transferase (MGST) is a membrane bound detoxification enzyme, which has been purified from different species including rat, mouse, human, frog, and fish. The rat MGST1 is localized predominantly in the liver microsomes and the outer mitochondrial membrane. The major function of the enzyme is conjugation of a broad range of hydrophobic xenobiotics with glutathione, which mostly results in detoxification and facilitates excretion. The activity of mammalian MGST1 (towards 1-chloro-2,4-dinitrobenzene, CDNB) could be activated upon the addition of sulfhydryl reagents (N-ethylmaleimide, NEM) and this interesting property has been studied continuously in our laboratory by experiments aimed at determining the kinetic (and functional) mechanism. Using alternate substrate diagnosis, the catalytic mechanism of the microsomal glutathione transferase 1 is defined as a random sequential mechanism. The activation of the enzyme, increasing the catalytic efficiency, was observed not only towards more reactive substances, but also towards less reactive substances such as 4-chloro-3nitrobenzamide when GSH is present at low concentration. The fact that the pKa of the thiol in the enzyme bound GSH (6.4) is lower than the pKa of free GSH (9.2) is an important aspect of the chemical catalysis. No significant change of the pKa value was found between unactivated and activated enzyme. 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 sulfonate) give hyperbolic binding isotherms with a stoichiometry of 1 mol per 1 mol of enzyme (i.e., 1 molecule bound per homotrimer). Glutathione had an equilibrium binding constant of 17 [my]M. Glutathione sulfonate was used in competition experiments and the results showed that glutathione sulfonate could effectively displace GSH. The Kd and Ki for glutathione sulfonate are both 10 [my]M. We found no change in these parameters between the unactivated and activated enzyme forms (by N-ethylmaleimide). Thus, we can conclude that activation does not result from changes in binding affinity to GSH. New microsomal glutathione transferases were purified from frog and pike liver. The two enzymes displayed extremely high activity towards 1-chloro-2,4-dinitrobenzene (200 times higher than vertebrate microsomal glutathione transferase 1 in unactivated form). Frog and pike liver MGST could be inactivated by NEM as a distinguishing property of the enzyme from lower vertebrates. The molecular mass of the two enzymes is about 17 kDa and the N-terminal amino acid sequence of pike microsomal glutathione transferase is similar to mammalian MGST1 The enzymes displayed glutathione peroxidase activity towards products of lipid peroxidation. Therefore, the suggested function of protection from lipid peroxidation was also identified in lower vertebrates. Traditional inhibitors of the MGST1 could also inhibit these enzymes. In contrast, the frog and pike enzymes displayed some differences in optimum pH range and inactivity towards N-acetyl-L-cysteine. The microsomal GST expressed in pike liver microsomes is estimated to be lower than 0.0022% of the total microsomal protein. This expression level is about 1400 times lower than that of rat liver microsomal GST1. So in lower vertebrates, microsomal GSTs displaying a lower expression level and a higher activity may be a balanced "strategy" in evolution. A simple rapid and sensitive fluorimetric method for the specific determination of reduced glutathione was developed. This method was based on glutathione S-transferase conjugation of GSH to monochlorobimane. Some important features such as the effects of dithioerythritol and the optimal conditions for forming the product GS-bimane were studied. The method is applicable to the determination of GSH in pure samples and in peripheral blood. A detection limit of 1 pmol was achieved. This assay is one of the most sensitive methods so far described. The method can thus be quite useful regarding research on the relationship between GSH levels and toxicity.
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