The binding of nitrate to the human anion exchange protein (AE1) studied with 14N nuclear magnetic resonance.
The binding of [14N]nitrate to the human erythrocyte anion transport protein, AE1, was studied using 14N nuclear magnetic resonance spectroscopy (14N-NMR). The line-width at half-height of the 14NO3- resonance increased in direct proportion to the concentration of erythrocyte ghost protein. Addition of the AE1 specific inhibitor 4,4'-dinitrostilbene-2,2'-disulfonate markedly reduced this line-broadening, indicating that the broadening was predominantly due to a specific interaction between nitrate and AE1. The dependence of the AE1 specific line-broadening on nitrate concentration had a first-order dissociation constant KD of 6.9 +/- 0.9 mM. In contrast, Cl- interaction with AE1 studied by 35Cl-NMR showed a chloride concentration-dependent line-broadening with a KD of 74 +/- 10 mM, indicating that AE1 has a higher affinity for nitrate than for chloride. Bicarbonate and chloride were found to be competitive inhibitors of the AE1 specific 14NO3- line-broadening (94 +/- 6% and 101 +/- 3% inhibition, respectively). Based on the concentration dependence of inhibition and using a model of competitive inhibition, the KD of bicarbonate binding to AE1 was estimated to be 5.4 +/- 1.3 mM. Nitrate is a structural analog of bicarbonate, making the interaction of nitrate with AE1 a good model for the bicarbonate-AE1 interaction. The 14N-NMR nitrate binding assay, along with the 35Cl-NMR binding assay now in use, will provide a powerful tool for studying the structure of the AE1 binding site for both physiologic substrates, bicarbonate and chloride.
Galanter
W L
WL
Department of Physiology, University of Illinois, Chicago 60680.
Labotka
R J
RJ
D001457:Anion Exchange Protein 1, Erythrocyte / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D001639:Bicarbonates / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D002712:Chlorides / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D009566:Nitrates / Q000378:metabolism*
D001457:Anion Exchange Protein 1, Erythrocyte; D001639:Bicarbonates; D002712:Chlorides; D009566:Nitrates