PHOSPHATE ANALOG PROBES OF RED CELL METABOLISM

基本上，红细胞膜上的所有阴离子运输都是 是由一种高度专门化的蛋白质“带3”完成的，负离子 运输蛋白，其生理功能是通过影响 氯化物与碳酸氢盐的极快速跨膜交换 在红细胞的呼吸周期中。这种蛋白质还可能 通过与质子的共同运输帮助调节细胞内pH 二价阴离子。我们建议使用P-31核磁共振来确定 光谱学可滴定物质输运的化学计量学 阴离子，即对总阴离子通量的贡献 单离子化(质子化)和双离子化(非质子化)形式 这些阴离子，以及这些阴离子可以在多大程度上共同运输其他 阳离子。这将通过比较质子通量来实现。 与阴离子交换有关的阴离子通量本身，用于 一系列具有不同pk值的可滴定磷酸盐类似物 以及截然不同的运输速度。此外，我们还将比较 含氮小分子输运的pH依赖性 不止一个阴离子基团(磷酸甲酸盐和 磷酸烯醇式丙酮酸)为含有相同大小的阴离子 只有一个阴离子基团(磷酸乙酯和乙酰磷酸酯)，到 确定运输站点中的功能组是否规范 这两类阴离子是不同的。两个这样的小阴离子， 乙酰磷酸盐(二价)和甲基乙酰磷酸盐 (单价)是能够乙酰化的反应性酸酐 氨基附近的反应。通过测量这些因素的影响 关于阴离子的电荷和结构特性的化合物 运输，我们将确定这些化合物是否可以修饰 功能阳离子残基在运输部位，我们认为 阴离子转运蛋白对以下阴离子具有极强的选择性 在结构上类似于碳酸氢盐，这是一种天然的底物 蛋白。我们将通过比较流入率来检验这一假设 对于一系列类碳酸氢盐阴离子，到化学上相关 不同结构的阴离子：亚硫酸盐-硫酸盐、亚磷酸盐- 磷酸盐、亚硒酸盐-亚硒酸盐和碲酸盐-碲酸盐。这些 将通过核磁共振波谱(P-31、Se-77、Te-125)进行测定， 比色法(亚硫酸盐、亚硒酸盐-亚硒酸盐)和原子比色法 吸收(碲酸盐-碲酸盐)。(Bi)亚硫酸盐类似于 以挥发酸(硫化氢)的阴离子形式存在的碳酸氢盐 它与S02和H20解离。我们将确定是否 中性二氧化硫在膜上的扩散有助于 对这种化合物的总通量有显著影响，以及 红细胞酶碳酸酐酶加速 这个过程通过催化亚硫酸盐和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.