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Molecular structure and functional mechanism of a novel proton pump H+-pyrophosphatase

新型质子泵H-焦磷酸酶的分子结构及作用机制

基本信息

批准号：
13142203
负责人：
MAESHIMA Masayoshi
金额：
$ 49.22万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2005
项目状态：
已结题

项目摘要

During the project period for five years, we obtained the following results concerning H+-pyrophosphatase and metal transporters.(1) H^+-pyrophosphatase (H^+-PPase) :H^+-PPase acidifies plant vacuoles or acidocalcisomes and maintains the pH gradient across the membrane. Our experiments revealed that a knockout mutant of H^+-PPase in Arabidopsis could not grow normally, suggesting that H^+-PPase is one of the essential membrane transport systems in plants.(2) Biochemical properties and membrane topology of H^+-PPase :In addition to plant H^+-PPase, we purified and characterized Streptomyces coelicolor H^+-PPase, since it and its mutant enzymes can be expressed in E. col. We revealed that S. coelicolor enzyme has a characteristic properties such as heat stability. We also determined the membrane topology of H^+-PPase and essential motifs in the enzyme. Our structural model has been accepted as the standard of H^+-PPases in various organisms.(3) Crystallization of H^+-PPase :We prepared a large quantity of Fv fragment, which specifically reacts with the active form of H+-PPase. We succeeded in preparation of a large quantity of the Fv-H+-PPase complex using the Fv fragment from the solubilized plant vacuolar membranes. Now, we are determining the best conditions for crystallization of the enzyme.(4) Cation/H^+ exchangers :Rice has five isoforms of cation/H^+ exchanger (CAX). These five isoforms varied with metal ion selectivity and tissue specific gene expression. CAXIa among them is expressed in all tissues examined and is highly expressed in companion and endodermis cells, which has high concentration of Ca^<2+>. We estimate that CAXla functions to reserve Ca^<2+> into vacuoles and is involved in the Ca^<2+> detoxification. The membrane topology and several functional motifs were also determined.

在五年的项目期间，我们获得了以下关于H+-焦磷酸酶和金属转运蛋白的结果。(1)H^+-焦磷酸酶（H^+-PPase）：H^+-PPase酸化植物液泡或酸钙体，并维持跨膜的pH梯度。我们的实验结果表明，H^+-PPase基因敲除突变体不能正常生长，表明H^+-PPase是植物必需的膜转运系统之一。(2)H^+-PPase的生物化学性质和膜拓扑结构：除了植物H^+-PPase外，我们还纯化和表征了天蓝色链霉菌H^+-PPase，因为它及其突变酶可以在E.我们发现S.腔肠酶具有诸如热稳定性的特性。我们还确定了H^+-PPase的膜拓扑结构和酶中的基本基序。我们的结构模型已被公认为各种生物中H^+-PP酶的标准。(3)H^+-PPase的结晶：我们制备了大量的Fv片段，其特异性地与H ^+-PPase的活性形式反应。利用植物液泡膜的可溶性片段成功地制备了大量的Fv-H ~+-PPase复合物。现在，我们正在确定酶结晶的最佳条件。(4)阳离子/H^+交换剂：水稻有五种阳离子/H^+交换剂（CAX）。这五种异构体因金属离子选择性和组织特异性基因表达而异。其中CAXIa在所有检测的组织中表达，并且在具有高浓度Ca^2+的伴胞和内皮细胞中高度表达。我们估计CAXla的功能是将Ca^<2+>储存到液泡中，并参与Ca^<2+>解毒。膜拓扑结构和几个功能基序也被确定。