COMPARATIVE PHYSIOLOGY OF MEMBRANE TRANSPORT

这项提案的长期目标是了解生理 和膜运输过程的功能的结构基础。的 具体目的是研究大肠杆菌的两种糖-阳离子共转运载体。杆菌 作为动物细胞中类似载体的“模型系统”（该实验室已 长期以来，他一直对葡萄糖-Na+共转运系统感兴趣， 小肠）。我们假设进化中最早的细胞 一个“质子经济”，使用质子泵和利用质子泵， 共运输过程的化学梯度。在动物进化的后期 细胞产生Na+-K+泵，Na+梯度用于共转运 流程.进化过程中可能的中间阶段的一个例子是 蜜二糖载体，可使用H+或Na+进行共转运。 一种实验方法将是确定 几种载体蛋白，以了解 不同的细胞类型乳糖载体或蜜二糖载体来自几个 微生物将被克隆和测序的桑格双脱氧核苷酸 法将在基因组的保守区域之间进行相关性分析。 转运蛋白和每个共同的生理参数。 第二种方法是测定载体中的氨基酸 蛋白质通常一方面识别糖，另一方面识别阳离子 另一边。糖识别和阳离子识别突变体将是 分离与几种技术，我们已经成功地利用在 过去载体蛋白的基因将被分离， 测序通过该方法发现的氨基酸取代的分析 应提供关于结合位点的有用信息。 第三种办法是研究联合国系统各部分的方向， 分子相对于膜的位置。既有遗传因素， 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.