Per Ålin

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.

Rat glutathione transferase 8-8, an enzyme efficiently detoxifying 4-hydroxyalk-2-enals

Rat glutathione transferase 8‐8 is one of the less abundant cytosolic glutathione transferases, accounting for approx. 1% of the total activity with 1‐chloro‐2,4‐dinitrobenzene in liver. The enzyme is eluted at pH 6.3 upon chromatofocusing and has so far been identified in liver, kidney, lung and testis. Characteristic properties include high relative activity with ethacrynic acid (70% of the specific activity with 1‐chloro‐2,4‐dinitrobenzene) and an apparent subunit M r of 24500. The most significant property noted is the high catalytic activity in the conjugation of 4‐hydroxyalk‐2‐enals, major products of lipid peroxidation. The catalytic efficiency with these substrates exceeds corresponding values for all known substrates tested with any glutathione transferase, which suggests that transferase 8‐8 may have evolved to detoxify 4‐hydroxyalk‐2‐enals.

Purification of major basic glutathione transferase isoenzymes from rat liver by use of affinity chromatography and fast protein liquid chromatofocusing

Seven major isoenzymes of glutathione transferase with isoelectric points ranging from pH 6.9 to 10 were isolated from rat liver cytosol. The purification procedure included affinity chromatography on immobilized S-hexylglutathione followed by high-performance liquid chromatofocusing. Characteristics, such as physical properties, reactions with antibodies, specific activities with various substrates, kinetic constants, and sensitivities to a set of inhibitors, are given for discrimination and identification of the different isoenzymes. The multiple forms of the enzyme correspond to glutathione transferases 1-1, 1-2, 2-2, 3-3, 3-4, and 4-4 in the recently introduced nomenclature [W.B. Jakoby et al. (1984) Biochem. Pharmacol. 33, 2539-2540]. A seventh form appears to be a heterodimeric protein composed of subunit 3 and an as yet unidentified subunit.

GLUTATHIONE TRANSFERASE FROM RAT TESTIS

Publisher Summary Glutathione transferases play an important role in the biotransformation and detoxication of electrophilic xenobiotics. The occurrence of glutathione transferase in animal species is widespread. Several transferase isoenzymes are isolated from rat liver. Six basic transferase isoenzymes in rat hepatic cytosol are characterized as binary combinations of four protein subunits. The presence of glutathione transferase is not restricted to the liver but also is demonstrated in extrahepatic organs. However, in comparison with the liver most other organs show considerably lower activity. One exception is rat testis, which also shows high transferase activity. In contrast with liver, and most other organs, a major part of the glutathione transferase activity in rat testis is borne by an isoenzyme, glutathione transferase 6-6, with an acidic isoelectric point. The purification of this enzyme, which accounts for approximately 50% of the cytosolic glutathione transferase activity, is described in this chapter. In addition to this major acidic isoenzyme, smaller amounts of the basic species, glutathione transferases 2-2, 3-3, 3-4, and 4-4 are identified in testis.

Structural evidence for three different types of glutathione transferase in human tissues

Cytosolic glutathione transferase was purified from human placenta and human liver. Three different forms of the enzyme were obtained, the acidic (π), the near‐neutral (μ), and the basic (α‐ϵ) forms; two had free α‐amino groups (π,μ) and one had a blocked α‐amino group (α‐ϵ). N‐terminal sequence analyses and total compositions gave clearly different results for each form, although transferases π and μ showed 35% sequence homology in the N‐terminal regions, with a 1‐residue shift in starting position. Consequently, the proteins are concluded to be products of three discrete but related genes.

[63] Glutathione transferase isoenzymes from rat liver cytosol

Publisher Summary This chapter describes the preparation of seven major glutathione transferases in rat liver cytosol. Six of these isoenzymes are formed as binary combinations of four different protein subunits resulting in different catalytic activities. An outline of the nomenclature of the glutathione transferases is also presented. The enzymatic activity is determined spectrophotometrically by measuring formation of the conjugate of the GSH and the CDNB at 340 nm. The purification procedure of the enzyme consists of several steps: preparation of microsome-free cytosol fraction, chromatography in sephadex G-25, affinity chromatography on S-hexylglutathione coupled to sepharose 6B, and chromatofocusing. The entire purification is performed at 5 ° . The procedure described in the chapter is dimensioned for one rat liver (7–10 g). It can readily be scaled up severalfold. Some of the molecular and catalytic characteristics of the six basic isoenzymes of glutathione transferase in rat liver cytosol are also reviewed.

Transformation of leukotriene A4 methyl ester to leukotriene C4 monomethyl ester by cytosolic rat glutathione transferases

Six major basic cytosolic glutathione transferases from rat liver catalyzed the conversion of leukotriene A4 methyl ester to the corresponding leukotriene C4 monomethyl ester. Glutathione transferasc 4‐4, the most active among these enzymes, had a V maxof 615 nmol · min−1 · mg protein−1 at 30°C in the presence of 5 mM glutathione. It was followed in efficiency by transferase 3–4 which had a V max of 160 nmol · min−1 · mg−1 under the same conditions. Transferases 1‐1, 1‐2, 2‐2 and 3‐3 had at least 30 times lower V max values than transferase 4‐4.

Glutathione Transferases Catalyzing Leukotriene C Synthesis and Metabolism of Leukotrienes C4 and E4 in Vivo and in Vitro

Glutathione transferases constitute a group of enzymes that catalyze several reactions involving glutathione (Mannervik, 1985). Their main function, according to current concepts, is to detoxify and accelerate the excretion of certain xenobiotic compounds (Chasseaud, 1979) by catalyzing the conjugation of glutathione with these electro-philic substrates. In addition, glutathione transferases also catalyze other reactions (e. g., peroxidase and isomerase reactions). We have recently reported (Mannervik et al., 1984) that six basic glutathione transferases from rat liver cytosol (Mannervik and Jensson, 1982) catalyze the conversion of LTA4 or its methyl ester to LTC4. One of the enzymes was a considerably more efficient catalyst of the reaction than the remaining five.

Species and tissue differences in the occurrence of isoenzymes of glutathione transferase

Glutathione transferase activity is widespread in biological species (see for instance Jakoby, 1978). Furthermore, it has been recognized that the enzyme usually occurs in multiple forms in an organism (Mannervik, 1984) and that several isoenzymes may exist in the same tissue. Within a species the isoenzyme pattern of an organ may be different in one individual from that of another, owing to genetic factors. Differences between individuals may also be due to enzyme induction. The underlying causes and the biological significance of the differences in the occurrence of isoenzymes of glutathione transferase are not fully understood. Nevertheless, the feature of isoenzymes has such prominence in the occurrence of glutathione transferase that it must be assumed to have great importance for the physiology of the organism. Investigation of structure, function, and occurrence of the various forms of glutathione transferase may consequently shed light on the biological roles of this important group of abundant proteins.