Studies on glutathione transport utilizing inside-out vesicles prepared from human erythrocytes.
Adenosine triphosphate-dependent glutathione transport was characterized using inside-out vesicles made from human erythrocytes. Kinetic analysis of the glutathione disulfide (GSSG) transport showed a biphasic Lineweaver-Burk plot as a function of GSSG concentration suggesting the operation of two different processes. One phase had a high affinity for GSSG and a low transport velocity. Most active at acidic pH and at 25 degrees C, this transport activity was easily lost during the storage of vesicles at 4 degrees C. The Km for Mg-ATP was 0.63 mM; guanosine triphosphate (GTP) substituted for ATP gave a 340% stimulation fo transport activity. Neither dithiothreitol nor thiol reagents affected this transport process. The other phase had a low affinity for GSSG and a high transport velocity. Most active at pH 7.2 and 37 degrees C, this transport activity was stable during storage of vesicles at 4 degrees C for several days. The Km for Mg-ATP was 1.25 mM; GTP substituted with no change in activity. Dithiothreitol increased the V but did not alter the Km, and thiol reagents inhibited the transport. These findings suggest that there are two independent transfer processes for GSSG in human erythrocytes.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001693:Biological Transport, Active; D004229:Dithiothreitol / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology
D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D005978:Glutathione; D008274:Magnesium; D004229:Dithiothreitol