MECHANISMS OF RENAL URATE TRANSPORT

所提出的研究旨在验证尿酸酶是一种 转运尿酸盐穿过肾血浆的膜相关蛋白 膜，并且至少两种转运体，阴离子交换剂和膜， 推定的转运蛋白尿酸酶存在于刷状缘和/或 肾近曲小管细胞的基底外侧膜 哺乳动物物种。 尿酸盐转运机制将在 未刺激和铜刺激的刷状缘和基底外侧膜 大鼠、犬和兔肾皮质的囊泡，用几种 技术：自由流电泳，镁聚集，钙 降水量和降水梯度。 运输将在 存在和不存在向内定向的电解质梯度，并且在 存在和不存在pH梯度。 动力学研究将评估 饱和、抑制和运输的温度依赖性。 的 在囊泡中运输的动力学常数将与那些 用于通过膜进行尿酸盐氧化。 为了进一步表征 尿酸盐转运机制、同交换和异交换现象 将对扩散进行评估。 细胞化学研究将在 肾和肝组织切片和肾膜囊泡定位 尿酸酶的膜内和细胞内位点。 该技术 基于氢与氢之间的耦合氧化-过氧化反应， 过氧化物，由尿酸盐的酶促（尿酸酶）氧化产生，以及铈 氯化 该反应的产物，过氧化铈， 在反应位点处的电子致密沉积物，从而允许 尿酸酶分布的电镜图。 物种 和肾膜，其中尿酸酶在转运中被检测， 细胞化学研究，尿酸酶将被分离和纯化。 溶解 膜将经受亲和层析。 纯化将 采用凝胶过滤、十二烷基硫酸钠（SDS）进行评估 聚丙烯酰胺凝胶电泳（PAGE）、梯度PAGE和等电 专注 纯化后，将产生多克隆抗体， 抗体对膜囊泡中尿酸盐转运的影响将是 考察 最后，将纯化的尿酸酶重组到人工合成的尿酸酶中， 缺乏尿酸酶的脂质膜或生物膜（红细胞 鬼）。 尿酸盐转运及转运和氧化的动力学常数 然后在重组系统中进行检查。 这些集体数据 应该为理解尿酸盐的作用机制提供坚实的基础。 运输，也许还有其他内源性和外源性物质的运输， 肾近端小管内的有机阴离子。

The proposed studies are designed to test the hypotheses that uricase is a membrane-associated protein that transports urate across renal plasma membranes, and that at least two transporters, an anion exchanger and the putative transporter, uricase, are present in brush border and/or basolateral membranes of the renal proximal tubule cells of several mammalian species. The mechanism(s) of urate transport will be assessed in unstimulated and copper stimulated brush border and basolateral membrane vesicles of rat, dog, and rabbit renal cortex prepared with several techniques: free-flow electrophoresis, magnesium aggregation, calcium precipitation, and percoll gradients. Transport will be examined in the presence and absence of inwardly directed electrolyte gradients, and in the presence and absence of pH gradients. Kinetic studies will assess saturation, inhibition and temperature dependence of transport. The kinetic constants of transport in vesicles will be compared to those obtained for urate oxidation by membranes. To further characterize the mechanism(s) of urate transport, the phenomena of homeo and heteroexchange diffusion will be evaluated. Cytochemical studies will be performed on renal and hepatic tissue slices and renal membrane vesicles to localize intramembranous and intracellular sites of uricase. This technique is based on a coupled oxidation-peroxidation reaction between hydrogen peroxide, produced by enzymatic (uricase) oxidation of urate, and cerium chloride. The product of this reaction, cerium perhydroxide, forms fine electron dense deposits at the site of the reaction, thereby permitting electron microscopic mapping of the distribution of uricase. In species and renal membranes in which uricase is detected in transport and cytochemical studies, uricase will be isolated and purified. Solubilized membranes will be subjected to affinity chromatography. Purification will be assessed with gel filtration, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), gradient PAGE, and isoelectric focusing. When purified, polyclonal antibodies will be raised and the effect of the antibodies on urate transport in membrane vesicles will be examined. Finally, purified uricase will be reconstituted into artificial lipid membranes or biological membranes devoid of uricase (red blood cell ghosts). Urate transport and kinetic constants of transport and oxidation will then be examined in the reconstituted system. This collective data should provide a solid base for understanding the mechanism(s) of urate transport, and perhaps the transport of other endogenous and exogenous organic anions within the renal proximal tubule.