BIOCHEMICAL STUDIES OF ATP-DRIVEN BACTERIAL TRANSPORT

Kdp是大肠杆菌的ATP驱动的钾转运系统。 它 由三个基因编码的三种蛋白质KdpA、KdpB和KdpC组成 kdpABC操纵子。 kdpABC操纵子的表达受 相邻kdpDE操纵子的两种产物的混合物。 KDP在功能上 类似于具有酰基磷酸中间体的转运ATP酶。 的 KdpB的序列与肌浆网的Ca-ATP酶同源。 主要重点仍然是遗传方法： 1.将kdpABC操纵子克隆在强的可控启动子后面。 2.分离、表征和测序改变但不改变的突变 取消KDP功能。 降低对K亲和力的突变在 研究;更多这样的突变将被调查。 突变， 主要影响是降低运输的Vmax也将进行研究。 3. KdpA已被鉴定为在形成K结合中具有主要作用。 KDP网站 KdpA的序列表明它穿过细胞膜， 次 将进行拓扑分析，以确定KdpA的哪些部分 暴露在内部，暴露在外部， 膜的 4.确定功能性Kdp复合物中的亚基相互作用。 5.鉴定和表征kdpDE操纵子的产物。 6.表征kdpABC启动子：对启动子区域进行测序， 确定转录起始位点。 7.将kdpDE克隆在强的可控启动子之后。 8.研究由kdpDE操纵子编码的蛋白质的性质;尝试 明确其在监管中的作用。 该项目得益于与Karlheinz Altendorf的合作 （西德国奥纳布吕克大学）几年。 这种合作 继续进行;主要或专门在奥斯纳尔布鲁克进行的工作，包括 9.纯化至Kdp的均一性。 10.测定KdpB的N-末端序列; KdpA、KdpB和KdpC的C-末端残基。

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.