Glutathione S-conjugate transport using inside-out vesicles from human erythrocytes

Previous studies [Kondo, T., Dale, G. L. and Beutler, E. (1981) Biochim. Biophys. Acta, 645, 132-136] have shown evidence for the existence of two different active-transport processes for glutathione disulphide (GSSG) in human erythrocytes (the high-Km and low-Km processes). In the present investigation adenosine-triphosphate-dependent transport of glutathione S-conjugate was characterized in comparison with active glutathione transport using inside-out vesicles from human erythrocytes. Incubation of the vesicles with glutathione S-conjugate (S-2,4-dinitrophenylglutathione) was found to inhibit competitively the high-Km process of GSSG transport but not significantly affect the low-Km process. The glutathione S-conjugate transport required ATP. A lineweaver-Burk plot of the transport rate as a function of the conjugate concentration gave an apparent Km value of 0.94 mM. The Km value of ATP-Mg was 0.76 mM. The transport of glutathione S-conjugate was dependent on temperature. Preincubation of vesicles with dithiothreitol resulted in an increase of the transport rate while thiol reagents, such as iodoacetamide, N-ethylmaleimide and p-chloromercuribenzoate inhibited the transport. Addition of nucleotides, such as CTP, UTP or GTP had no effect on the transport. These findings suggest that glutathione S-conjugate formed by the catalytic reaction of glutathione S-transferase in erythrocytes under the exposure to electrophilic compounds, is eliminated via the same transport process for GSSG elevated under oxidative stress.