The role of thiols in ATP-dependent transport of S-(2,4-dinitrophenyl)glutathione by rat liver plasma membrane vesicles.

The effect of thiol/disulfide exchange on ATP-dependent S-(2,4-dinitrophenyl)glutathione (GS-DNP) transport was studied in sodium nitrate treated rat liver plasma membrane vesicles. Transport followed Michaelis-Menten kinetics with an apparent Km of 9.6 microM for GS-DNP and 124 microM for ATP. 5,5'-Dithiobis(2-nitrobenzoate) (DTNB) and N-ethylmaleimide (NEM) efficiently inactivated GS-DNP transport activity in a dose- and time-dependent manner. Half-maximal inactivation occurred in 10 min at 40 microM for DTNB and 550 microM for NEM. Inactivation by DTNB was reversed by dithiothreitol. S-(N-Ethyl)maleimyl glutathione and/or ATP-Mg2+, but neither S-(N-ethyl)maleimyl cysteinylglycine nor oxidized glutathione could protect transport activity from inactivation by NEM or cystamine. These results suggest that reactive thiols are located near the active site of the transporter and that S-alkyl and the gamma-glutamyl residues of glutathione are important for protection. Biological disulfides which were tested included cystine, oxidized glutathione, oxidized Coenzyme-A, oxidized lipoic acid, and oxidized lipoamide; cystamine was the most potent reversible inactivator. Molecular oxygen also inactivated transport activity, which was recovered on addition of dithiothreitol, suggesting intramolecular disulfide formation by vicinal thiols. We interpret these results to indicate that the ATP-dependent GS-DNP transporter contains two or more thiols which are necessary for the maintenance of transport activity. The reversible inactivation of the activity by biological disulfides suggests that the transporter may be regulated by thiol/disulfide exchange in vivo.