Helgi Jensson

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.

Selective expression of glutathione transferase isoenzymes in chemically induced preneoplastic rat heptocyte nodules

Isoenzymes of glutathione transferase were shown to occur at selectively altered levels in rat hepatocyte nodules produced by 2‐acetylaminofluorene treatment. Changes were measured by different substrates, antibodies raised against purified glutathione transferases, and by purification of the major isoenzymes. Isoenzymes composed of subunits 1, 2 and 3, expressed in normal liver tissue, all occurred at increased concentrations in nodules, whereas the level of transferase 4‐4 was decreased. The most conspicuous change was the appearance of glutathione transferase 7‐7 (or transferase P), the concentration of which in negligible in normal liver.

A set of inhibitors for discrimination between the basic isozymes of glutathione transferase in rat liver

A set of inhibitors including hematin, bromosulfophthalein, and triethyltin bromide was used for discrimination and identification of the major basic isozymes of glutathione transferase in rat liver cytosol. Six enzymes are formed as binary combinations of 4 protein subunits: A, B, C, and L. Discrimination between the transferases can be based on the differences of the subunits in susceptibility to the inhibitors. The identification of transferase subunits is further supported by the combined use of specific substrates and inhibitors.

[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.