BIOCHEMICAL STUDIES OF ATP-DRIVEN BACTERIAL TRANSPORT

KDP是一种由ATP驱动的大肠杆菌钾转运系统。它 由三个基因编码的蛋白KdpA、KdpB和KdpC组成 KdpABC操纵子的。KdpABC操纵子的表达受到控制 相邻kdpDE操纵子的两个产物。KDP在功能上 类似于用酰基磷酸中间体运输ATPase。这个 KdpB的序列与肌浆网的Ca-ATPase同源。 主要的重点仍然是遗传方法： 1.将kdpABC操纵子克隆到一个强大、可控的启动子后面。 2.分离、鉴定和测序可改变但不改变的突变 取消KDP功能。降低K亲和力的突变正在进行中 研究；将对更多这样的突变进行调查。突变，其 主要作用是降低Vmax的运输也将进行研究。 3.KdpA已被确定在形成K结合的过程中起主要作用 KDP的站点。KdpA的序列表明它穿过了许多细胞膜 泰晤士报。将执行拓扑图以确定KdpA的哪些部分 内部暴露，外部暴露，并在 薄膜。 4.确定功能KDP复合体中的亚基相互作用。 5.鉴定和鉴定kdpDE操纵子的产物。 6.鉴定kdpABC启动子：测序启动子区域和 确定转录起始位置。 7.在一个强大、可控的启动子背后克隆kdpDE。 8.研究由kdpDE操纵子编码的蛋白质的性质；尝试 确定它们在监管中的角色。 这个项目得益于与卡尔海因茨·阿尔滕多夫的合作 (大学)西德奥纳布吕克)已有数年。此协作 继续；主要或仅在奥斯纳布鲁克完成的工作包括 9.对KDP进行纯化，使其达到均一。 10.KdpB N-末端序列的测定； KdpA、KdpB和KdpC的C端残基。

Kdp is an ATP-driven potassium transport system of Escherichia coli. It consists of three proteins, KdpA, KdpB and KdpC, encoded by the three genes of the kdpABC operon. Expression of the kdpABC operon is under the control of the two products of the adjacent kdpDE operon. Kdp is functionally similar to transport ATPases with an acyl-phosphate intermediate. The sequence of KdpB is homologous to the Ca-ATPase of sarcoplasmic reticulum. Major emphasis remains on genetic approaches: 1. Clone kdpABC operon behind a strong, controllable promoter. 2. Isolate, characterize and sequence mutations which alter but do not abolish Kdp function. Mutations which reduce affinity for K are under study; many more such mutations will be investigated. Mutations whose primary effect is to reduce the Vmax of transport will also be studied. 3. KdpA has been identified as having a major role in forming the K binding site of Kdp. The sequence of KdpA suggests it crosses the membrane many times. Topological will be performed to identify which parts of KdpA are exposed internally, which are exposed externally and which are in the membrane. 4. Determine subunit interactions in the functional Kdp complex. 5. Identify and characterize the products of the kdpDE operon. 6. Characterize the kdpABC promoter: sequence the promoter region and determine the site of transcription initiation. 7. Clone kdpDE behind a strong, controllable promoter. 8. Study properties of the proteins encoded by the kdpDE operon; attempt to define their roles in regulation. This project has beniffted from collaboration with Karlheinz Altendorf (Univ. of O nabruck, West Germany) for several years. This collaboration continues; work to be done mainly or exclusively in Osnarbruck, includes 9. Purification to homogeneity of Kdp. 10. Determination of the N-terminal sequence of KdpB; determination of the C-terminal residues of KdpA, KdpB and KdpC.