Essentially all anion transport across the red cell membrane is accomplished by a highly specialized protein, """"""""Band 3"""""""", the Anion Transport Protein, which functions physiologically by effecting the extremely rapid transmembrane exchange of chloride for bicarbonate in the respiratory cycle of the erythrocyte. This protein may also help regulate intracellular pH by cotransport of protons with divalent anions. We propose to determine using P-31 NMR spectroscopy the stoicheiometry of the transport of titratable anions, that is, the contributions to total anion flux by the monoionized (protonated) and di-ionized (unprotonated) forms of these anions, and to what extent these anions can cotransport other cations. This will be accomplished by comparing the proton fluxes associated with anion exchange to the anion fluxes themselves, for a series of titratable phosphate analogs having different pK values and widely different transport rates. In addition, we will compare the pH dependence of the transport of small molecules containing more than one anionic group (phosphonoformate and phosphoenolpyruvate) to that for anions of similar size containing only one anionic group (ethyl phosphate and acetyl phosphate), to determine whether functional groups in the transport site regulate these two classes of anions differently. Two such small anions, acetyl phosphate (divalent) and methyl acetyl phosphate (monovalent) are reactive anhydrides capable of acetylation reactions near amino groups. By measuring the effects of these compounds on the charge and structural specificity of anion transport, we will determine whether these compounds can modify functional cationic residues in the transport site, we believe that the anion transport protein is extremely selective for anions that structurally resemble bicarbonate, the natural substrate for this protein. We will test this hypothesis by comparing influx rates for a series of bicarbonate-like anions, to chemically related anions of different structure: sulfite-sulfate, phosphite- phosphate, selenite-selenate and tellurite-tellurate. These determinations will be by NMR spectroscopy (P-31, Se-77, Te-125), colorimetric methods (sulfite, selenite-selenate) and atomic absorption (tellurite-tellurate). (Bi)sulfite is similar to bicarbonate in being the anionic form of a volatile acid (H2S03) which dissociates to S02 and H20. We will determine whether diffusion of neutral S02 across the membrane contributes significantly to the total flux of this compound, and whether the erythrocytic enzyme carbonic anhydrase accelerates this process by catalyzing the interconversion of sulfite and sulfur dioxide.
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