Studies on hepatic glutathione transferases from rat and man : 1. A kinetic mechanism for glutathione transferase A from rat liver. 2. Transferase µ, a new form of glutathione transferase in human liver

Sammanfattning: This study of glutathione transferases was undertaken to achieve a better understanding of the catalytic and molecular properties of various isozymes of this group of drug-metabolizing enzymes.The steady-state kinetics of purified glutathione transferase A from rat liver with glutathione (GSH) and 1,2-dichloro- 4-nitrobenzene as substrates, did not follow a simple Michaelis- Menten equation. Inhibition by the product of the enzymatic reaction, as well as by other S-substituted glutathione derivatives, was also nonlinear. The complex kinetics observed were shown not to be the result of partial inhibition of the enzyme by ethanol (a solvent present in the assay system).Binding studies showed that glutathione transferase A can bind GSH, bromosulfophthalein, the product of the enzymatic reaction, or glutathione disulfide with a maximal binding stoichiometry of 2 molecules per protein dimer. No sign of cooperativity was observed.The experimental data of the kinetic and binding studies were analyzed by nonlinear regression analysis.A steady-state sequential mechanism, in which the two substrates bind and the two products leave in random order, is proposed for glutathione transferase A.The isozyme patterns of glutathione transferase in different human adult and fetal livers were investigated by means of isoelectric focusing. All fetal livers contained a basic and an acidic transferase as major constituents. In contrast, multiple basic forms and only minor amounts of acidic transferases were found in adult liver. Some individuals possessed an additional previously unknown glutathione transferase isozyme, with a near-neutral isoelectric point.This new isozyme, transferase µ, was purified to apparent homogeneity. Affinity chromatography on S-hexylglutathione Sepharose 6B was a very efficient step in this purification.The molecular and catalytic properties of transferase µ are distinct from those of the other human transferases, which indicates that transferase µ is a separate gene product. Of particular interest is the high activity of transferase µ towards the epoxides benzo(a)pyrene 4,5-oxide and styrene 7,8- oxide. transferase µ also binds non-substrate ligands, e.g., cholate and bilirubin.