William H. Habig

The purification of homogeneous glutathione S-transferases B and C from rat liver is described. Kinetic and physical properties of these enzymes are compared with those of homogeneous transferases A and E. The letter designations for the transferases are based on the reverse order of elution from carboxymethylcellulose, the purification step in which the transferases are separated from each other. Transferase B was purified on the basis of its ability to conjugate iodomethane with glutathione, whereas transferase C was purified on the basis of conjugation with 1,2-dichloro-4-nitrobenzene. Although each of the four enzymes can be identified by its reactivity with specific substrates, all of the enzymes are active to differing degrees in the conjugation of glutathione with p-nitrobenzyl chloride. Assay conditions for a variety of substrates are included. All four glutathione transferases have a molecular weight of 45,000 and are dissociable into subunits of approximately 25,000 daltons. Despite the similar physical properties and overlapping substrate specificities of these enzymes, only transferases A and C are immunologically related.

Evidence is presented that ligandin, an intracellular protein involved in the binding of such anions as bilirubin, indocyanine green, and penicillin, is identical to glutathione S -transferase B (EC 2.5.1.18), an enzyme catalyzing the conjugation of glutathione with such electrophiles as 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, iodomethane, ethacrynic acid, and bromosulfophthalein. The proteins, isolated by distinct methods, have the same specificity for substrates and for ligands, react in identical fashion to antibody produced against ligandin, bear entirely similar physical characteristics and amino acid composition, and are both induced in response to phenobarbital. Indocyanine green, one of the ligands that is not effective as a substrate, was shown to competitively inhibit the conjugation reaction. It is suggested that specificity is directed toward compounds with electrophilic sites.

Glutathione transferase A has been purified from rat liver. The enzyme catalyzes the conjugation of glutathione with compounds bearing an electrophilic site, especially those in which the electrophilic site is on, or α to, an aromatic ring. The enzyme has a molecular weight of 45,000 and is composed of two similar subunits. Initial velocity, product inhibition, and binding studies indicate a biphasic kinetic mechanism in which the reaction pathway depends on the concentration of the substrates. At high concentrations of GSH, an ordered sequential pathway predominates in which GSH binds first. At low concentrations of GSH, a ping-pong pathway predominates in which the electrophilic substrate adds first. In accordance with a prediction of the general rate equation for the over-all mechanism, the breakpoint in the biphasic double reciprocal plot for GSH saturation was found to shift to lower GSH concentrations as the concentration of the electrophilic substrate was lowered. A numerical rate equation was developed which describes initial velocities over the entire range of substrate concentrations. An appendix presents a method for distinguishing among several formal kinetic mechanisms which yield nonlinear double reciprocal initial velocity plots as a result of multiple reaction pathways.