COMPARATIVE PHYSIOLOGY OF MEMBRANE TRANSPORT

这项提议的长期目标是理解生理上的 以及膜转运过程功能的结构基础。这个 具体目的是研究大肠杆菌的两种糖阳离子共转运载体 作为动物细胞中类似载体的“模型系统”(本实验室有 长期以来一直对人的葡萄糖-Na+共转运系统感兴趣 小肠)。我们假设进化中最早的细胞利用 “质子经济”，使用质子泵和利用质子电子-- 共输送过程的热化学梯度。在后来的进化中动物 细胞进化出Na~+-K~+泵，Na~+梯度用于共转运 流程。进化中可能处于中间阶段的一个例子是 一种可以使用H+或Na+进行共运输的二糖载体。 一种实验方法将是确定氨基酸序列 几种载体蛋白看载体之间的关系 不同的细胞类型。乳糖载体或蜂蜜糖载体 微生物将被Sanger双脱氧核苷酸克隆和测序 方法。将在两个基因的保守区之间建立相互关系 运输蛋白和每个共同的生理参数。 第二种方法是确定载体中的氨基酸。 通常一方面识别糖和阳离子的蛋白质 在另一端。糖识别和阳离子识别突变体将是 我们已经成功地使用了几种技术 过去时。载体蛋白的基因将被分离，DNA 已排序。用这种方法发现的氨基酸取代的分析 应提供有关结合位点的有用信息。 第三种方法是研究不同细分市场的定位 相对于膜的分子。既有遗传的，也有 将使用免疫学方法。

The long-term objective of this proposal is to understand the physiological and structural basis for the function of membrane transport processes. The specific aim is to study two sugar-cation cotransport carriers of E. coli as "model system" for similar carriers in animal cells (this laboratory has had a longstanding interest in the glucose-Na+ cotransport system of the small intestine). We postulate that the earliest cell in evolution utilized a "proton economy", using proton pumps and utilizing the proton elec- trochemical gradient for cotransport processes. Later in evolution animal cells evolved Na+-K+ pumps and the Na+ gradient was used for cotransport processes. One example of a possible intermediate stage in evolution is the melibiose carrier which can use either H+ or Na+, for cotransport. One experimental approach will be to determine the amino acid sequence of several carrier proteins to see the relationship between carriers of different cell types. The lactose carrier or melibiose carrier from several microorganisms will be cloned and sequenced by the Sanger dideoxynucleotide method. A correlation will be made between the conserved regions of the transport protein and the physiological parameters common to each. A second approach will be to determine the amino acids in the carrier protein that normally recognize the sugar on the one hand and the cation on the other. Sugar recognition and cation recognition mutants will be isolated with several techniques which we have successfully utilized in the past. The gene for the carrier protein will be isolated and the DNA sequenced. Analysis of the amino acid substitutions found by this method should provide useful information concerning the binding sites. A third approach will be a study of the orientation of various segments of the molecule with respect to the membrane. Both genetic as well as immunological methods will be utilized.