The major isoenzymes of cytosolic glutathione transferase (EC 2.5.1.18) from rat, mouse, and man are shown to share structural and catalytic properties that can be used for species-independent classification. Rat, mouse, and human isoenzymes were grouped with respect to aminoterminal amino acid sequences, after correlation of seven structures analyzed in the present investigation with structures determined earlier. The isoenzymes were also characterized by substrate specificities and sensitivities to inhibitors, and the data were subjected to pattern recognition analysis. In addition, the various isoenzymes were tested for cross-reactivity by immunoprecipitation with antibodies raised against rat and human transferases. The different types of data were clearly correlated and afforded an unambiguous division of the isoenzymes into three classes named alpha, mu, and pi. Each of the three mammalian species studied contains at least one isoenzyme of each class. It is suggested that the similarities of the isoenzymes in a class reflect evolutionary relationships and that the classification applies generally.

Three distinct glutathione transferases in the liver cytosol fraction of male NMRI mice have been purified by affinity chromatography and fast protein liquid chromatofocusing. These enzymes account for approximately 95% of the activity detectable with 1-chloro-2,4-dinitrobenzene as electrophilic substrate. Differences between the three forms are manifested in isoelectric points, apparent subunit molecular mass values, amino acid compositions, N-terminal structures, substrate specificities, and sensitivities to inhibitors, as well as in reactions with specific antibodies raised against glutathione transferases from rat and human tissues. The results indicate strongly that the three mouse enzymes are products of different genes. A comparison of the mouse glutathione transferases with rat and human enzymes revealed similarities between the transferases from different species. Mouse glutathione transferases have been named on the basis of their respective subunit compositions.

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.

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The enzyme-catalysed conjugation of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+/-)-anti-BPDE] with glutathione (GSH) by cytosolic GSH transferases isolated primarily from rat lung has been studied. GSH transferase 4-4 was active in the GSH conjugation of anti-BPDE, whereas transferases 2-2 and 3-3 showed little activity. GSH transferase 1-1 did not contribute to the activity since significant amounts were not detected in the rat lung. Activity was also obtained with several acidic pulmonary GSH transferases and with a newly described form, transferase 7-7, also isolated from rat kidney and from hyperplastic liver nodules. The catalytic efficiency (kcat/Km) of transferase 7-7 was seven times that of transferase 4-4, the most active rat transferase previously identified. When the GSH concentration was varied at constant (+/-)-anti-BPDE concentration in the presence of transferases 4-4, 7-7 or the major acidic transferase, non-linear Lineweaver-Burk plots were obtained. Resolution of the GSH conjugates of the two enantiomers of (+/-)-anti-BPDE by h.p.l.c. showed that all isoenzymes with notable activity were selective (greater than or equal to 97%) for the (+)-enantiomer of anti-BPDE, which is generally considered to be the most carcinogenic form of BPDE. The possibility that one enantiomer inhibits the conjugation of the other enantiomer with GSH cannot be excluded and may quantitatively affect the results obtained.