The relation between membrane structure and NADH: (Acceptor) oxidoreductase activity of erythrocyte ghosts

Abstract

Human red blood cell ghosts (RBCG) prepared by gradual osmotic lysis (Danon procedure) down to 30 mOsm (G-30) were exposed to a 9 mOsm hypotonic treatment (G-9) which increased NADH-ferricyanide reductase activity by 272% and the (Mg + Na + K) ATPase activity by 40%. Sonic oscillation of G-30 did not affect ATPase but increased NADH oxidation 7 times. After fragmentation the entire NADH ferricyanide activity remained bound to membrane particles and showed the same specificity for electron acceptors and donors. The effect of temperature and divalent ions on the NADH oxidation was also studied in intact arid fragmented ghosts. The results indicate that permeability to the substrates and the vectorial assembly of active sites of the enzyme are the rate limiting factors of this reaction. Histochemical observation of the erythrocyte ghosts, using tetrazolium salt (NBT) as electron acceptor, allowed us to locate the enzyme on the membrane. The effect of detergents, phospholipases A and C, and organic solvents on the NADH: (acceptor)oxidoreductase and ATPase showed: (a) complete inactivation of both enzymes at 0.5% sodium dodecyl sulphate; (b) phospholipases A and C inhibited (Mg + Na + K)ATPase but not NADH oxidation; (c) treatment of freeze-dried membrane particles with anhydrous organic solvents such as acetone, pentane or butanol did not affect either of two enzymic activities, but a gradual inactivation occurred upon aqueous-acetone treatment. The NADH: (acceptor)oxidoreductase was dissociated from the phospholipidprotein complex by an aqueous butanol extraction after the removal of loosely bound lipid with anhydrous butanol. These results indicate that NADH: (acceptor)oxidoreductase of RBCG as well as (Mg + Na + K)-ATPase is a structural enzyme of these membranes.