BINDING OF GLUTATHIONE AND AN INHIBITOR TO MICROSOMAL GLUTATHIONE TRANSFERASE

Microsomal glutathione transferase is an abundant liver protein that c an be activated by thiol reagents. It is not known whether the activat ion is associated with changed binding properties of the enzyme. There fore the binding of GSH and an inhibitor to rat liver microsomal gluta thione transferase was studied by use of equilibrium dialysis and equi librium partition in a two-phase system. The radioactive substrate glu tathione and an inhibitor (glutathione sulphonate) give hyperbolic bin ding isotherms with a stoichiometry of 1 mol per mol of enzyme (i.e. 1 molecule per homotrimer). Glutathione had an equilibrium binding cons tant of 18 mu M. Competition experiments involving glutathione sulphon ate showed that it could effectively displace GSH. These and kinetic s tudies showed that the K-d and K-i for glutathione sulphonic acid are close to 10 mu M. No change in these parameters was obtained after N-e thylmaleimide activation of the enzyme. Thus activation does not resul t from changes in binding affinity to GSH.