Selective phenylglyoxalation of functionally essential arginyl residues in the erythrocyte anion transport protein.

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Abstract
The red cell anion transport protein, band 3, can be selectively modified with phenylglyoxal, which modifies arginyl residues (arg) in proteins, usually with a phenylglyoxal:arg stoichiometry of 2:1. Indiscriminate modification of all arginine in red cell membrane proteins occurred rapidly when both extra- and intracellular pH were > 10. Selective modification of extracellularly exposed arginine was achieved when ghosts with a neutral or acid intracellular pH were treated with phenylglyoxal in an alkaline medium. The rate and specificity of modification depend on the extracellular chloride concentration. At 165 mM chloride maximum transport inactivation was accompanied by the binding of 4 phenylglyoxal/band 3 molecule. After removal of extracellular chloride, maximum transport inhibition was accompanied by the incorporation of 2 phenylglyoxals/band 3, which suggests that transport function is inactivated by the modification of a single arginine. After cleavage of band 3 with extracellular chymotrypsin, [14C]phenylglyoxal was located almost exclusively in a 35,000-dalton peptide. The primary covalent binding site of the isothiocyanostilbenedisulfonates is a lysyl residue in the 2nd cleavage product, a 65,000-dalton fragment. The transport region of band 3 apparently is composed of strands from both chymotryptic fragments. The binding of phenylgloxal and the stilbene inhibitors interfered with each other. The rate of phenylglyoxal binding was reduced by reversibly binding stilbenedisulfonate (DNDS), and covalent binding of [3H]DIDS [4,4''-diisothiocyanostilbene-2,2''-disulfonic acid] to phenylglyoxal-modified membranes was strongly delayed. At DIDS concentrations below 10 .mu.M, only 50% of the band 3 molecules were labeled with [3H]DIDS during 90 min at 38.degree. C, thereby demonstrating an interaction between binding of the 2 inhibitors to the protomers of the oligomeric band 3 molecules.

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