MEMBRANES AND ACTIVE TRANSPORT OF AMINO ACIDS

膜和氨基酸的主动运输

基本信息

批准号：
3150817
负责人：
GIOVANNA F AMES
金额：
$ 23.19万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
1977
资助国家：
美国
起止时间：
1977-01-01 至 1985-12-31
项目状态：
已结题

项目摘要

This project is concerned with defining mechanisms by which substances are transported into and out of cells and with analyzing the biosynthesis and structure of bacterial membranes. The experiments will make use of microorganisms (Salmonella typhimurium) which can be manipulated genetically by isolating mutants defective in the various steps of active transport or of membrane biogenesis, which can then be analyzed biochemically. The histidine transport system, which we use as a modely system, is composed of three proteins: the histidine-binding protein J (periplasmic, already characterized), the P protein (membrane bound) and the Q protein. The P protein will be purified and characterized, which is an essential step in the understanding of the molecular mechanism of transport. The Q protein will be identified, purified, and characterized. To aid us in these efforts, we have cloned the histidine transport operon and will be able to identify missing components and produce large amounts of protein for purification purposes. The clone will also be used for sequencing the DNA of these genes in order to understand their regulation, the secretion mechanism of membrane and periplasmic proteins. We will attempt to reconstitute an active transport system in liposomes or spheroplasts. We will research the interaction of periplasmic proteins with membranes by cross-linking experiments. We are also starting a study of the energy coupling mechanisms functioning in our system: we essentially plan to test the hypothesis that acetylphosphate is the energy source for periplasmic systems (Hong) by isolating appropriate mutants in acetylphosphate biosynthsis and by assaying directly the level of acetylphosphate in the cell. The multiplicity of function of the membrane-bound component, P protein, will be investigated by studying its involvement with a lysine-, arginine-, ornithine-binding protein in vivo and in vitro.

该项目涉及定义物质的机制转运进出细胞并分析生物合成和细菌膜的结构。实验将利用可以通过基因操作的微生物（鼠伤寒沙门氏菌）分离在主动运输的各个步骤中存在缺陷的突变体或膜生物发生，然后可以进行生化分析。组氨酸我们用作模型系统的运输系统由三个部分组成蛋白质：组氨酸结合蛋白 J（周质，已表征）， P 蛋白（膜结合）和 Q 蛋白。 P蛋白将是纯化和表征，这是理解的重要一步运输的分子机制。 Q蛋白将被鉴定，纯化并表征。为了帮助我们开展这些工作，我们克隆了组氨酸转运操纵子，将能够识别缺失的成分和产生大量用于纯化目的的蛋白质。克隆人还将用于对这些基因的 DNA 进行测序，以了解它们的调节，膜和周质蛋白的分泌机制。我们将尝试在脂质体中重建主动运输系统或原生质球。我们将研究周质蛋白与通过交联实验形成膜。我们也开始研究能量耦合机制在我们的系统中发挥作用：我们基本上计划检验乙酰磷酸是周质能量来源的假设系统（Hong）通过在乙酰磷酸生物合成中分离适当的突变体并直接测定细胞中乙酰磷酸的水平。这膜结合成分 P 蛋白的功能多样性将是通过研究其与赖氨酸、精氨酸的参与进行调查，体内和体外鸟氨酸结合蛋白。

项目成果

期刊论文数量（16）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Analysis of promoter mutations in the histidine transport operon of Salmonella typhimurium: use of hybrid M13 bacteriophages for cloning, transformation, and sequencing.

鼠伤寒沙门氏菌组氨酸转运操纵子启动子突变分析：使用杂交 M13 噬菌体进行克隆、转化和测序。

DOI：
10.1128/jb.159.3.1000-1005.1984
发表时间：
1984
期刊：
Journal of bacteriology
影响因子：
3.2
作者：
Lee,GS;Ames,GF
通讯作者：
Ames,GF

Regulatory regions of two transport operons under nitrogen control: nucleotide sequences.

氮控制下两个转运操纵子的调节区：核苷酸序列。

DOI：
10.1073/pnas.79.4.1083
发表时间：
1982
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Higgins,CF;Ames,GF
通讯作者：
Ames,GF

Physical map of the Salmonella typhimurium histidine transport operon: correlation with the genetic map.

鼠伤寒沙门氏菌组氨酸转运操纵子的物理图谱：与遗传图谱的相关性。