Glutathione turnover in human erythrocytes. Inhibition by buthionine sulfoximine and incorporation of glycine by exchange.

Previous investigations have indicated that [`4C]glycine is incorporated into erythrocyte glutathione at a rate which exceeds by severalfold the rate of cysteine and glutamate incorporation. The discrepancy between the rates has generally been attributed to the known differences in the rates at which these amino acids are transported into erythrocytes. In the present investigation, human erythrocytes were incubated with ['4C]glutamate or [14C]glycine, and the speciific activities of the intracellular amino acid and glutathione pools were determined at intervals for 6 h. The experiments with [`4C]glycine indicate an apparent glutathione turnover time of about 1 day, whereas those with [14C]glutamate indicate a turnover time of about 6 days. Since the calculations are based on the specific activities of the intracellular amino acid pools, the discrepancy between the two turnover rates cannot be attributed to differences in the rates of amino acid transport. It is concluded that the results with [14C]glutamate reflect the true rate of glutathione biosynthesis, and that glycine is incorporated into erythrocyte glutathione primarily by an exchange reaction catalyzed by glutathione synthetase. In support of this conclusion, de novo glutathione biosynthesis as measured with [14C]glutamate or [35Sjcystine is inhibited about 90% by buthionine sulfoximine, an inhibitor of -y-glutamylcysteine synthetase; glycine incorporation, in contrast, is inhibited <10% by buthionine sulfoximine. It is apparent that glutathione turnover cannot be meaningfully measured with radioactive glycine in erythrocytes or, by inference, in other tissues with high levels of glutathione synthetase or slow rates of glutathione biosynthesis.