Membrane protein complexes in GSH-depleted red cells

G6PD-deficient red cells (nonhemolytic variants) incubated for 24 hr in medium containing glucose maintain their adenosine triphosphate (ATP) levels but show a pronounced fall in reduced glutathione (GSH) levels. Membranes of such ATP-replete, GSH-depleted red cells contain a high molecular weight protein aggregate that is disulphide bonded. This aggregate is similar in composition to that found in control red cells that are incubated in the absence of substrate (i.e., ATP- and GSH-depleted) in that considerable amounts of spectrin are present with a high spectrin:band 3 ratio. These cells also show a marked decrease in spectrin “extractability” and do not show any overt shape change on phase-contrast microscopy. Addition of acetylphenylhydrazine to the medium results in a high molecular weight aggregate consisting primarily of globin, although such cells are also ATP-replete and GSH-depleted. Our findings indicate that spectrin rearrangement can occur in ATP-replete red cells, but that such cells can still maintain their discoid shape. Addition of autologous plasma or albumin to substrate-containing medium in which G6PD-deficient cells are incubated has a marked protective effect on membrane protein aggregate formation. This effect is mediated across a dialysis membrane. Although the exact mechanism is unclear, the protective effect of albumin does not appear to be mediated by scavenging ambient oxidants or binding trace amounts of metals. These in vitro studies support recent concepts of mechanisms of hemolysis in hemolytic G6PD variants in which normal red cell ATP levels and spectrin-containing membrane aggregates have been detected.