Molecular Characterization of Vacuolar H+-Translocating Inorganic Pyrophosphatase

ATP在初级离子转运中的主导作用 是普遍接受的。 阳离子转位ATP酶，能够 泵H+，Na+，K+和Ca 2+，已被发现在几乎所有 活细胞，并被认为是至关重要的， 细胞功能 然而，一个值得注意的例外是H+- 易位无机焦磷酸酶（H+-PPase） 植物液泡膜（液泡膜），利用无机 焦磷酸（PPi）代替ATP作为能量来源。 这 酶，PPi依赖性H+泵的第一个例子，在任何“非- 能量耦合”膜，是液泡膜的主要组成部分 并且能够产生H+-电化学电势 差+200mV。 因此，液泡膜PPase 在蛋白质和功能上都具有定量意义， 水平并适合分子表征。 本研究提供了（i）氨基的测定 Mr 64，000底物结合亚基的氨基酸序列 （二）组织的组成成分; 功能集成泵及其说明 各自的角色。 目标（一）深入了解 这种新的易位酶的进化起源，并促进 在膜上模拟其组织。 目标㈡，关于 另一方面，提供了一个独立的测试模型 来源于序列数据，并有助于识别 结构性 负责介导底物水解和 H+易位。 植物液泡膜PPase的普遍性 细胞和PPi的独特状态作为高浓度的限制性情况， 能量磷酸盐将使获得的结果广泛 生物能量学意义 高等植物细胞的液泡膜（液泡膜）含有 一种叫做H+转移无机焦磷酸酶的酶。 这种酶在高等植物中普遍存在，并有助于 跨膜电化学反应器建立与维护 电位差 这种电化学电势差， 反过来，对于其他溶质的运输是必需的。 以来 对这种重要的酶知之甚少，研究正在进行中。 以了解其进化起源， 组织和生理功能。

The primacy of ATP for the energization of primary ion transport is universally accepted. Cation-translocating ATPases, capable of pumping H+, Na+, K+ and Ca2+, have been found in virtually all living cells and are recognized to be of paramount importance for cellular function. One notable exception, however, is the H+- translocating inorganic pyrophosphatase (H+-PPase) of higher plant vacuolar membrane (tonoplast) which utilizes inorganic pyrophosphate (PPi), instead of ATP, as energy source. This enzyme, the first example of a PPi-dependent H+ pump in any "non- energy coupling" membrane, is a major vacuolar membrane component and capable of generating a H+-electrochemical potential difference of +200mV. The tonoplast PPase is therefore quantitatively significant at both the protein and functional levels and amenable to molecular characterization. This research provides for (i) the determination of the amino acid sequence of the Mr64,000 substrate-binding subunit of the enzyme, and (ii) the enumeration of the components constituting the functionally integral pump and elucidation of their respective roles. Objective (i) provides insights into the evolutionary origins of this novel translocase and facilitate the modeling of its organization on the membrane. Objective (ii), on the other hand, provides an independent test of the models derived from the sequence data and facilitate identification of the structural elements responsible for mediating both substrate hydrolysis and H+-translocation. The ubiquity of the tonoplast PPase in plant cells and the unique status of PPi as the limiting case of a high energy phosphate will make the results obtained of broad bioenergetic significance. The vacuolar membrane (tonoplast) of higher plant cells contains an enzyme called a H+-translocating inorganic pyrophosphatase. This enzyme is ubiquitous in higher plants and contributes to the establishment and maintenance of a transmembrane electrochemical potential difference. This electrochemical potential difference, in turn, is necessary for the transport of other solutes. Since little is known about this important enzyme, studies are being carried out to obtain knowlegde of its evolutionary origins, organization and physiological functions.