Towards the localization of the essential arginine residues in the band 3 protein of human red blood cell membranes.
The effects of 4-hydroxy-3-nitrophenylglyoxal (HNPG), on the binding of eosin-5-maleimide (EMA), and diethyl pyrocarbonate (DEPC) to the anion transport system in the human red blood cell membrane, have been investigated. HNPG is a reversibly binding, arginine-specific, anion transport competitive inhibitor, known to act on the anion binding site. The EMA reaction site is an external facing lysine residue (Lys-430) in the 17 kDa transmembrane segment. The DEPC reaction site is an intracellular histidine (His-819) in the 35 kDa fragment. The results show that inhibition of the transport system with EMA increases in presence of HNPG to about 2.3 times. This finding suggests a positive cooperativity between the HNPG and EMA binding site and give evidence that the essential arginine is either nearby or allosterically linked to Lys-430. The inhibition of the cells with DEPC was nearly unchanged or slightly decreased in the presence of 10 mM HNPG. These results suggest that the intracellular His-residue which reacts with DEPC is not a part of the transport pathway. Our experiments with 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) have shown that its affinity to the transport system does not change after pre-treatment with phenylglyoxal (PG). We also found that the binding of [14C]phenylglyoxal (PG) to band 3 reduces significantly in presence of chloride. This is another evidence for the direct involvement of arginine residues in substrate binding.
Böhm
R
R
Max-Planck-Institut für Biophysik, Frankfurt am Main, Germany.
Zaki
L
L
D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry / Q000378:metabolism*; D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D002250:Carbon Radioisotopes; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D010658:Phenylglyoxal / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D013267:Stilbenes / Q000737:chemistry
D001457:Anion Exchange Protein 1, Erythrocyte; D002250:Carbon Radioisotopes; D013267:Stilbenes; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid; D001120:Arginine; D004047:Diethyl Pyrocarbonate; D010658:Phenylglyoxal