Bengt Mannervik

Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver.

Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver have been investigated. After perfusing the lung to remove contaminating blood, this organ was found to have an apparent concentration of glutathione (2mM) which is approx. 20% of that found in the liver. Both organs contain very low levels of glutathione disulfide. Neither phenobarbital nor methylcholanthrene had a significant effect on the levels of reduced glutathione in lung and liver. In addition, the activities of some glutathione-metabolizing enzymes--glutathione reductase and glutathione S-transferase activity assayed with four different substrates--were observed to be 5-to 60-fold lower in lung tissue than in the liver.

[59] Glutathione reductase

Publisher Summary Glutathione reductase is a flavoprotein catalyzing the NADPH-dependent reduction of glutathione disulfide (GSSG) to glutathione (GSH). The reaction is essential for the maintenance of glutathione levels. Glutathione has a major role as a reductant in oxidation–reduction processes, and serves in detoxication and several other cellular functions of great importance. A purification method of this enzyme from calf liver and rat liver is described in this chapter. Similar methods are used for the purification of the enzyme from yeast, porcine, and human erythrocytes. All the steps are carried out at about 5 ° . The purification method from calf liver consists of various steps including preparation of cytosol fraction, chromatography on DEAE-sephadex, precipitation with ammonium sulfate, and chromatography on hydroxyapatite. The purification of glutathione reductase from rat liver is usually combined with the preparation of glutathione transferases, thioltransferase, and glyoxalase I.

[28] Glutathione transferase (human placenta)

Publisher Summary Glutathione transferases with basic isoelectric points have been purified from the human liver, and an acidic form is purified from human erythrocytes. However, this chapter presents a procedure for the preparation of glutathione transferase from human placenta. Enzyme activity during purification is determined spectrophotometrically at 340 nm by measuring the formation of the conjugate of glutathione and l-chloro-2,4-dinitrobenzene. Purification procedure involve the extraction from the human placenta; the supernatant fraction from Step 1 is chromatographed on a column (12.5 x 80 cm) of Sephadex G-25 and the pooled fractions from Step 2 are applied to a column (9 x 13 cm) of DEAE-cellulose equilibrated with l0 mM Tris-HCl at pH 7.8. The pooled effluent from Step 3 is dialyzed overnight against 3 x 10 liter of 10 mM sodium phosphate buffer; then affinity chromatography is carried on S-hexylglutathione coupled to epoxy-activated sepharose 6B and the pooled fractions from Step 5 are charged onto a column of Sephadex G-75 that is equilibrated and eluted with 10 mM sodium phosphate at pH 6.7, containing 0.1 mM dithioerythritol. The active fractions of effluent are pooled.

4‐Hydroxyalk‐2‐enals are substrates for glutathione transferase

The 4‐hydroxyalk‐2‐enals are established products of lipid peroxidation that are conjugated with intracellular glutathione. Cytosolic glutathione transferases from rat liver were shown to give high specific activities with 4‐hydroxynonenal and 4‐hydroxydecenal. The isoenzyme giving the highest specific activity was glutathione transferase 4‐4. The rate of the spontaneous conjugation reaction is negligible in comparison with the rate calculated for the cellular concentration of the glutathione transferases. It is proposed that a major biological function of the glutathione transferases is to protect the cell against products of oxidative metabolism, such as epoxides, organic hydroperoxides, and 4‐hydroxyalkenals.

Nomenclature for Mammalian Soluble Glutathione Transferases

The nomenclature for human soluble glutathione transferases (GSTs) is extended to include new members of the GST superfamily that have been discovered, sequenced, and shown to be expressed. The GST nomenclature is based on primary structure similarities and the division of GSTs into classes of more closely related sequences. The classes are designated by the names of the Greek letters: Alpha, Mu, Pi, etc., abbreviated in Roman capitals: A, M, P, and so on. (The Greek characters should not be used.) Class members are distinguished by Arabic numerals and the native dimeric protein structures are named according to their subunit composition (e.g., GST A1-2 is the enzyme composed of subunits 1 and 2 in the Alpha class). Soluble GSTs from other mammalian species can be classified in the same manner as the human enzymes, and this chapter presents the application of the nomenclature to the rat and mouse GSTs.

[62] Glutathione transferases from human liver

Publisher Summary This chapter investigates glutathione transferases derived from human liver. The glutathione transferases are a group of related enzymes that catalyze the conjugation of glutathione with a variety of hydrophobic compounds bearing an electrophilic center. The proteins also act as intracellular binding proteins for a large number of lipophilic substances, including bilirubin. Human glutathione transferases have been purified from liver, erythrocytes, placenta, and lung. A simple and rapid procedure for the purification of basic (α-ɛ) and neutral (μ) glutathione transferases from human liver cytosol is described in the chapter. The enzyme activity during purification is determined spectrophotometrically at 340 nm by measuring the formation of the conjugate of glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB). The steps of the purification procedure include (1) preparation of cytosol fraction, (2) chromatography on Sephadex G-25, (3) chromatography on DEAE-cellulose, and (4) chromatography on Sephadex G-25.

[60] Glutathione peroxidase

Publisher Summary Glutathione peroxidases catalyze the reduction of hydroperoxides (ROOH) by glutathione (GSH). “R” may be an aliphatic or aromatic organic group or, simply, hydrogen. The products are H 2 O, an alcohol (ROH), and glutathione disulfide (GSSG). Regeneration of GSH from GSSG in the cell is effected by the enzyme glutathione reductase. Assays of glutathione peroxidase activity are based on the measurement of ROOH or GSH consumption. Alternatively, GSSG production is monitored by coupling to the reaction catalyzed by glutathione reductase. Oxidation of NADPH is recorded spectrophotometrically or fluorometrically. This chapter provides an overview of the assay methods of glutathione peroxidase enzyme. There are two major types of glutathione peroxidase. One type is distinguished by containing selenium in the form of covalently bound selenocysteine in its active site. This selenium-dependent enzyme is active with both organic hydroperoxides and H 2 0 2 . The second type of glutathione peroxidase consists of proteins that do not depend on selenium for catalysis and have negligible activity with H 2 0 2 . This class is constituted by glutathione transferases.

Crystal structure of human glyoxalase I--evidence for gene duplication and 3D domain swapping.

The zinc metalloenzyme glyoxalase I catalyses the glutathione‐dependent inactivation of toxic methylglyoxal. The structure of the dimeric human enzyme in complex with S‐benzyl‐glutathione has been determined by multiple isomorphous replacement (MIR) and refined at 2.2 Å resolution. Each monomer consists of two domains. Despite only low sequence homology between them, these domains are structurally equivalent and appear to have arisen by a gene duplication. On the other hand, there is no structural homology to the ‘glutathione binding domain’ found in other glutathione‐linked proteins. 3D domain swapping of the N‐ and C‐terminal domains has resulted in the active site being situated in the dimer interface, with the inhibitor and essential zinc ion interacting with side chains from both subunits. Two structurally equivalent residues from each domain contribute to a square pyramidal coordination of the zinc ion, rarely seen in zinc enzymes. Comparison of glyoxalase I with other known structures shows the enzyme to belong to a new structural family which includes the Fe2+‐dependent dihydroxybiphenyl dioxygenase and the bleomycin resistance protein. This structural family appears to allow members to form with or without domain swapping.

[21] Regression analysis, experimental error, and statistical criteria in the design and analysis of experiments for discrimination between rival kinetic models

Publisher Summary The fitting of rate equations to kinetic data in enzymology is an application of the treatment of experimental data in general and the use of mathematical models for quantitative description. By using statistical methods, a certain degree of objectivity is ascertained insofar as all investigators should get the same analytical results once they have agreed on the techniques to use. Certain statistical fitting procedures also provide quantitative measures of goodness of fit and of the reliability of the kinetic constants estimated, facilitating evaluation of the results and testing of hypotheses. This is the case for nonlinear regression analysis based on the principle of least squares. In this chapter, it is assumed that a mathematical model (rate equation) should be fitted by nonlinear regression analysis to a set of experimental data. Most procedures use the principle of least squares. While discrimination between rival mathematical models, two questions arise: do the models adequately describe the data? Is one model better than the other is? The first question may be answered by evaluating the results of the regression by the criteria for goodness of fit. If both models are adequate and fit the data equally well, the simplest model is chosen. However, independent information obtained by additional kinetic studies or by completely different experimental methods should be included in the discrimination procedure.

Crystal structure of human glyoxalase II and its complex with a glutathione thiolester substrate analogue.

BACKGROUND Glyoxalase II, the second of two enzymes in the glyoxalase system, is a thiolesterase that catalyses the hydrolysis of S-D-lactoylglutathione to form glutathione and D-lactic acid. RESULTS The structure of human glyoxalase II was solved initially by single isomorphous replacement with anomalous scattering and refined at a resolution of 1.9 A. The enzyme consists of two domains. The first domain folds into a four-layered beta sandwich, similar to that seen in the metallo-beta-lactamases. The second domain is predominantly alpha-helical. The active site contains a binuclear zinc-binding site and a substrate-binding site extending over the domain interface. The model contains acetate and cacodylate in the active site. A second complex was derived from crystals soaked in a solution containing the slow substrate, S-(N-hydroxy-N-bromophenylcarbamoyl)glutathione. This complex was refined at a resolution of 1.45 A. It contains the added ligand in one molecule of the asymmetric unit and glutathione in the other. CONCLUSIONS The arrangement of ligands around the zinc ions includes a water molecule, presumably in the form of a hydroxide ion, coordinated to both metal ions. This hydroxide ion is situated 2.9 A from the carbonyl carbon of the substrate in such a position that it could act as the nucleophile during catalysis. The reaction mechanism may also have implications for the action of metallo-beta-lactamases.