Glutathione S‐Conjugate Transport Using Inside‐Out Vesicles from Human Erythrocytes

Abstract

Previous studies [Kondo, T., Dale, G. L. and Beutler, E. (1981) Biochim. Biophys. Acta, 645, 132‐1361 have shown evidence for the existence of two different active‐transport processes for glutathione disulphide (GSSG) in human erythrocytes (the high‐K_m and low‐K_m 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‐K_m process of GSSG transport but not significantly affect the low‐K_m 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 K_m value of 0.94 mM. The K_m 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 andp‐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.