Anion Exchange in Bacteria

本研究考察了革兰氏阴性厌氧菌formigenes草酸杆菌中发现的一种新型交换蛋白的物理和细胞生化。在这种厌氧菌中，二价草酸盐与单价甲酸盐的交换形成了质子动力产生的基础。由于其在细胞生物化学中的作用，这种反port反应在“化学渗透”载体中是前所未有的，在阴离子交换蛋白中因其致电特性而独特。已经设计了一些实验来重建这种不损失功能的反端口反应，并用作从O. formigenes膜中纯化交换蛋白的指导。在蛋白质纯化后，实验将揭示动力学反应机制（乒乓或同步）和反应循环的一部分，在膜上携带电荷。利用残基特异性化学探针，制备针对该反端口蛋白的单克隆抗体，并对其物理结构进行氨基酸序列预测。草酸杆菌是一种革兰氏阴性厌氧菌，其代谢能量的唯一来源是草酸的脱羧。已经确定，O. formigenes的膜含有一个载体，显示二价草酸盐和一价甲酸盐的电交换。细胞生物化学对这种反应特别感兴趣，因为它是生物体维持质子动力的一种机制。因此，前体和产物的反端口导致负电荷的净进入，而草酸脱羧过程中质子的消耗改变了内部ph。这是这种“二级”载体的新用途，并且确定其他利用羧酸的细胞是否也利用这种反端口反应是有趣的。本研究将探讨该载体的分子特性及其在细胞生物化学中的整体功能。这项工作将有助于目前对细菌如何组织其细胞功能的理解。

This research examines the physical and cellular biochemistry of a novel exchange protein found in the gram-negative anaerobe Oxalobacter formigenes. In this anaerobe, the exchange of divalent oxalate for monovalent formate forms the basis for generation of proton-motive force. This antiport reaction is unprecedented among "chemiosmotic" carriers for its role in cellular biochemistry and unique among anion exchange proteins for its electrogenic character. Assays have been devised to reconstitute this antiport reaction without loss of function and are used as a guide to purify the exchange protein from membranes of O. formigenes. After the protein is purified experiments will reveal the kinetic reaction mechanism (Ping Pong or Simultaneous) and that portion of the reaction cycle which carries charge over the membrane. Using residue-specific chemical probes, monoclonal antibodies directed against the antiport protein, and the derived amino acid sequence predictions will be made about the physical structure. Oxalobacter formigenes, a gram-negative anaerobe, is a cell whose sole source of metabolic energy derives from the decarboxylation of oxalic acid. It has been established that membranes of O. formigenes contain a carrier that displays the electrogenic exchange of divalent oxalate and monovalent formate. This reaction is of particular interest to cellular biochemistry as a mechanism whereby the organism sustains a proton-motive force. Thus, the antiport of precursor and product results in the net entry of negative charge, while consumption of a proton during oxalate decarboxylation changes internal pH. This is a novel use for such a "secondary" carrier and is of interest to determine whether other cells that utilize carboxylic acids also exploit such antiport reactions. This research will examine the molecular properties of this carrier and its overall function in cellular biochemistry. This work will contribute to the current understanding of how bacteria organize their cellular functions.