Mechanism of inhibition of glutathione efflux by methionine from isolated rat hepatocytes.

We studied mechanism of inhibition of glutathione (GSH) efflux by methionine with freshly isolated rat hepatocytes. Inhibition was specific for L-methionine and was not due to changes in membrane potential or cell volume. Methionine (100 microM) inhibited GSH efflux from cells having 20-60 nmol GSH/10(6) cells. Inhibition was overcome in cells with greater than 75 nmol GSH/10(6) cells. Kinetics of control and inhibited efflux were sigmoidal saturable and were fitted well with the Hill model having three cooperative binding per transport sites. Vmax was the same for both cases (0.24 +/- 0.013 nmol X min-1 X 10(6) cells-1), implying that the inhibition was competitive. Apparent Km of efflux was 3.3 +/- 0.20 mM for controls but was shifted to 5.6 +/- 0.14 mM (P less than 0.01) in the presence of 100 microM methionine. Kinetic analysis of the inhibition by varying concentrations of methionine estimated Ki = 61.3 +/- 6.0 microM and n = 1.2 +/- 0.07, suggesting involvement of a single inhibition site. Methionine uptake was independent of GSH concentration, and blocking its uptake with 2-amino-2-norbornanecarboxylic acid did not affect inhibition. When methionine-preloaded cells were resuspended in methionine-free buffer, GSH efflux rapidly returned to control levels, whereas digitonin-releasable cellular methionine remained nearly constant. Thus, inhibition appeared to be exerted from outside the cell, possibly through an allosteric mechanism. A consequence of inhibition of GSH efflux by methionine was decreased uptake of cysteine equivalents from extracellular cystine.