Reversible and irreversible inhibition of phosphate transport in human erythrocytes by a membrane impermeant carbodiimide.

Abstract

Phosphate entry into chloride-loaded human erythrocytes is inhibited by treatment of cells with the water-soluble carbodiimide 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) in the absence of added nucleophile. EAC does not penetrate the erythrocyte membrane or lead to significant intermolecular cross-linking of membrane proteins. At neutral extracellular pH in chloride-free medium, only about 50% of transport is rapidly and irreversibly inhibited, but at alkaline pH, inhibition is more rapid and complete. Inhibition by EAC was reversible in the presence of extracellular NaCl. Modification of membrane sulfhydryl groups does not prevent inhibition of phosphate transport by EAC but almost complete protection is afforded by 4,4-dinitrostilbene-2,2-disulfonic acid, a reversible competitive inhibitor of anion transport. N-(4-Azido-2-nitrophenyl)-2-aminoethylsulfonate, a reversible noncompetitive inhibitor of anion transport did not protect against EAC inhibition of transport but prevented reversal of inhibition in saline medium. Transport inhibition by [3H]EAC did not lead to specific incorporation of radioactivity into Band 3, the anion transport protein. These results suggest that inhibition of anion transport by EAC is due to modification of a carboxylic acid residue in or near the transport site accessible from the external face of the membrane. The subsequent fate of the modified carboxyl residue appears to be sensitive to the orientation of the anion transport site.