PHOSPHATE ANALOG PROBES OF RED CELL METABOLISM

基本上所有阴离子通过红细胞膜的转运都是 由一种高度专业化的蛋白质，“带3”，负离子， 转运蛋白，其生理功能是通过影响 氯化物对碳酸氢盐的极快跨膜交换 在红细胞的呼吸循环中。 这种蛋白质也可以 通过质子共转运帮助调节细胞内pH 二价阴离子。 我们建议使用P-31 NMR测定 光谱学可滴定物质传输的化学计量学 阴离子，也就是说，总阴离子通量的贡献， 单电离（质子化）和双电离（非质子化）形式的 这些阴离子，以及在何种程度上这些阴离子可以共转运其他 阳离子。 这将通过比较质子通量 与阴离子交换相关的阴离子通量本身， 一系列具有不同pK值的可滴定磷酸盐类似物 和不同的传输速率。 此外，我们将比较 pH依赖性的小分子的运输， 多于一个阴离子基团（膦酰基甲酸酯和 磷酸烯醇式丙酮酸）与类似大小的阴离子相比， 仅一个阴离子基团（磷酸乙酯和乙酰基磷酸）， 确定转运位点中的官能团是否调节 这两类阴离子的不同。 两个如此小的阴离子， 乙酰磷酸（二价）和乙酰磷酸甲酯 （单价）是能够乙酰化的反应性酸酐 氨基附近的反应。 通过测量这些影响， 阴离子的电荷和结构特异性 运输，我们将确定这些化合物是否可以改变 在转运位点的功能性阳离子残基，我们认为， 阴离子转运蛋白对阴离子具有极高选择性， 结构上类似于碳酸氢盐，天然底物， 蛋白 我们将通过比较流入率来检验这一假设 对于一系列类似碳酸氢根的阴离子， 不同结构的阴离子：亚硫酸根-硫酸根，亚磷酸根- 磷酸盐、亚硒酸盐-硒酸盐和亚碲酸盐-碲酸盐。 这些 将通过NMR光谱法（P-31、Se-77、Te-125）进行测定， 比色法（亚硫酸盐、亚硒酸盐-硒酸盐）和原子吸收法 吸收（碲酸盐-碲酸盐）。 （Bi）亚硫酸盐类似于 碳酸氢盐是挥发性酸（H2SO 3）的阴离子形式 其解离成SO2和H2O。 我们将决定 中性SO2穿过膜的扩散有助于 显着的总通量的这种化合物，以及是否 红细胞碳酸酐酶 该方法通过催化亚硫酸盐的相互转化， 二氧化硫

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.