MEMBRANES AND ACTIVE TRANSPORT OF AMINO ACIDS

希替丁在鼠伤寒沙门氏菌和大肠杆菌中的转运 Coli需要4种蛋白质：组氨酸结合蛋白、周质蛋白、J和 3个膜结合组分，Q、M和P。 蛋白质已经被完全测序。P蛋白已被证明含有 一种核苷酸结合部位，被认为与 运输中的能量耦合。J，Q，M， 用交联法和亲和标记法对P进行分析 试剂，从膜的两边进行蛋白质分解消化， 无膜试剂的放射性标记。膜泡 将通过添加J来重组以用于运输并用于研究 渗透酶的能量偶联和膜结构。这个 将对P蛋白的核苷酸结合部位进行表征，并 将寻求酶功能(如ATP水解)。这个 膜结合的成分将被提纯，生化表征和 用于囊泡和脂质体的重建实验。为了这个 目的，针对每种蛋白质的抗体将通过以下方式制备 使用β-半乳糖苷酶融合蛋白或通过各种其他 接近了。全细胞和囊泡中的能量耦合机制 将会被探究，因为关于 能量耦合的性质(PMF或底物水平的磷酸化) 周质系统。预计通过使用彻底的 特征的周质系统(如组氨酸渗透酶)和井 UNC(ATPase)和负责基因的突变体的特征 生产乙酰磷酸盐(ACK和PTA)应该可以清除 这场冲突。将寻求一种强有力的遗传方法来帮助 理解能量耦合。因为所有的周质系统似乎 同样地，不相关系统之间的关系也将被组织起来 通过计算机分析和基因技术进行探索；取代 具有来自无关系统的组件的有缺陷的传输组件 将通过基因选择。运输操作员的监管和 将探索邻近的argt基因对氮素有效性的影响。 将建立调节性NTRC蛋白的DNA结合位点 通过滤膜结合试验和足迹法检测。我们的假设是NTRC 可能起到抗终止剂作用的蛋白质将通过确定 在NTRC在场和不在场的情况下记录的终止地点 蛋白质，包括体内和体外。氮素调控突变体将是 通过体内和体外重组DNA诱变产生。

The transport of hidtidine in Salmonella typhiphimurium and Escherichia coli requires 4 proteins: a histidine-binding, periplasmic protein, J, and 3 membrane-bound components, Q, M, and P. The operon which codes for these proteins has been fully sequenced. The P protein has been shown to contain a nucleotide-binding site which is postulated to be involved in energy-coupling in transport. The architectural organization of J, Q, M, and P will be analyzed by the use of cross-linking and affinity labelling reagents, proteolytic digestion from either side of the membrane, radioactive labelling with membrane-impermeant reagents. Membrane vesicles will be reconstituted for transport by addition of J and used for studying the energy-coupling and the membrane organization of the permease. The nucleotide-binding site of the P protein will be characterized and an enzymatic function (such as ATP hydrolysis) will be sought. The membrane-bound components will be purified, characterized biochemically and used for reconstitution experiments in vesicles and liposomes. For this purpose, antibodies to each of the proteins will be prepared, either by using Beta-galactosidase-fusion proteins or by a variety of other approaches. The mechanism of energy coupling in whole cells and vesicles will be explored since there is considerable confusion concerning the nature of energy coupling (pmf or substrate-level phosphorylation) for periplasmic systems. It is expected that by using a thoroughly characterized periplasmic system (such as the histidine permease) and well characterized mutants in unc (ATPase) and in genes responsible for producing acetylphosphate (ack and pta) it should be possible to clear up this conflict. A strong genetic approach will be pursued to help understand energy coupling. Since all periplasmic systems seem to be similarly organized the relationship between unrelated systems will be explored by computer analysis and by genetic techniques; replacement of a defective transport component with a component from an unrelated system will be selected genetically. The regulation of the transport operon and of the neighboring argT gene by nitrogen availability will be explored. The DNA-binding sites for the regulatory ntrC protein will be established by filter-binding assay and footprinting. Our hypothesis that the ntrC protein might act as an antiterminator will be tested by determining the termination site of transcripts in the presence and absence of the ntrC protein, both in vivo and in vitro. Nitrogen regulatory mutants will be created both in vivo and by in vitro recombinant DNA mutagenesis.