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A highly purified plasma membrane fraction isolated from rat hepatocytes was found to catalyze the hydrolysis of ATP in response to micromolar concentrations of glutathione disulfide (GSSG). This process exhibited distinct kinetic parameters suggesting the existence of both a high and low affinity component. The apparent Km values (GSSG) for ATP hydrolysis were 140 microM and 1 mM for the high affinity and low affinity components, respectively. Disulfides other than GSSG were also found to stimulate ATP hydrolysis. The similarity between the kinetic properties of the GSSG-stimulated ATPase and those reported for GSSG transport in erythrocytes (Kondo, T., Dale, G. L., and Beutler, E. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6359-6362) suggests that the ATPase may function in the active extrusion of intracellular GSSG.

The intracellular distribution of glutathione (GSH) in cultured hepatocytes has been investigated by using the compound monochlorobimane (BmCl), which interacts specifically with GSH to form a highly fluorescent adduct. Image analysis of BmCl-labeled hepatocytes predominantly localized the fluorescence in the nucleus; the nuclear/cytoplasmic concentration gradient was approximately three. This concentration gradient was collapsed by treatment of the cells with ATP-depleting agents. The uneven distribution of BmCl fluorescence was not attributable to (i) nonspecific interaction of BmCl with protein sulfhydryl groups, (ii) any selective nuclear localization of the GSH transferase(s) catalyzing formation of the GSH-BmCl conjugate, or (iii) any apparent alterations in cell morphology from culture conditions, suggesting that this distribution did, indeed, reflect a nuclear compartmentalization of GSH. That the nuclear pool of GSH was found more resistant to depletion by several agents than the cytoplasmic pool supports the assumption that GSH is essential in protecting DNA and other nuclear structures from chemical injury.