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A large number of human tumor cell lines of various origins have been investigated with respect to expression of glutathione-linked enzymes in the cytosol fraction. The amounts of the different enzymes were estimated by use of activity measurements and by silver staining or immunoblot analysis after electrophoresis of cytosol fractions purified by affinity chromatography on S-hexylglutathione Sepharose. Class Pi glutathione transferase was the most abundant enzyme in most tumor cells; the cell lines HepG2 and Raji were exceptions in not expressing significant amounts of this enzyme. HepG2 cells derive from hepatocytes, which normally do not express the class Pi enzyme, whereas Raji cells originate from B-lymphocytes, which normally do express a class Pi glutathione transferase. The highest level of the class Pi transferase, in terms of protein reacting with antibodies as well as enzyme activity, was noted in the colon carcinoma cell line LS174T. Hu549Pat cells, EBV-transformed B-lymphocytes, also expressed high levels of a protein reacting with antibodies specific for class Pi glutathione transferases, but did not display any significant activity with ethacrynic acid, a substrate characteristic for this class. Class Alpha and class Mu glutathione transferases, in cell lines expressing these isoenzymes, were present in significantly lower concentrations than the class Pi enzyme. Most of the tumor cells contained a class Alpha transferase composed of 27.5 kd subunits, which has the physicochemical and immunological properties of the most basic glutathione transferase found in human skin. In several cell lines, a protein was detected with an apparent subunit Mr value of 30 kd that was tentatively identified as an additional class Alpha glutathione transferase not previously described. In addition, other glutathione-linked enzyme activities, namely glutathione peroxidase, glutathione reductase and glyoxalase I, were assayed with specific substrates in the cytosolic fraction of the tumor cells; glyoxalase I could also be estimated semiquantitatively by silver staining of SDS-PAGE cells after affinity chromatography. Like the glutathione transferases, these enzymes displayed distinctly different levels of expression in the various cell lines. Thus, virtually every cell line was found to have a unique pattern of glutathione-linked enzymes, suggesting that the resistance phenotypes of the cells differ accordingly.