Isolation and characterization of the multiple glutathione S-transferases from human liver. Evidence for unique heme-binding sites.
Thirteen forms of glutathione S-transferase were isolated from human liver in high yields by glutathione-affinity chromatography and chromatofocusing. Apparent isoelectric points ranged from 4.9 to 8.9 and included neutral forms. All 13 forms appeared to be identical immunochemically in a quantitative enzyme-linked immunosorbent assay. These forms were immunochemically distinct from the major acidic glutathione S-transferase found in placenta and erythrocyte and were immunochemically distinct from two forms of higher molecular weight glutathione S-transferase found in some but not all liver samples. The 13 forms exhibited similar activities with 1-chloro-2,4-dinitro-benzene as substrate, specific activities of 33-94 mumol/min/mg. Likewise, these forms all exhibited glutathione peroxidase activity with cumene hydroperoxide, specific activities of 1.5-8.3 mumol/min/mg. All 13 forms bound bilirubin with subsequent conformational changes leading to states devoid of transferase activity, a process prevented by the presence of foreign proteins. As hematin-binding proteins, however, these multiple transferases exhibited a very broad range of binding extending from nonbinding to high-affinity binding (KD approximately 10(-8) M). Hematin binding was noncompetitive with transferase activity and did not involve the bilirubin-binding site, suggesting the existence of unique heme-binding sites on these proteins. The two forms of the immunochemically distinct glutathione S-transferases transferases found in some liver samples also exhibited both transferase and peroxidase activities. In addition, they also have separate sites for binding bilirubin and hematin.
D000818:Animals; D001663:Bilirubin / Q000378:metabolism; D001665:Binding Sites; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004912:Erythrocytes / Q000201:enzymology; D005260:Female; D005982:Glutathione Transferase / Q000302:isolation & purification* / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D006427:Hemin / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D010920:Placenta / Q000201:enzymology; D011817:Rabbits
D006418:Heme; D006427:Hemin; D005982:Glutathione Transferase; D001663:Bilirubin