Molecular Characterization of Vacuolar H+-Translocating Inorganic Pyrophosphatase

人们普遍认为，三磷酸腺苷对初级离子传输的带电作用是首要的。阳离子转位ATPase能够泵出H+、Na+、K+和Ca~(2+)，几乎在所有活细胞中都被发现，并被认为对细胞功能至关重要。然而，一个值得注意的例外是高等植物液泡膜(液泡膜)的H+-转运型无机焦磷酸酶(H+-PPase)，它利用无机焦磷酸(PPI)而不是ATP作为能源。这种酶是任何“非能量耦合”膜中依赖PPI的H+泵的第一个例子，是液泡膜的主要成分，能够产生+200 mV的H+-电化学电位差。因此，液泡膜PPase在蛋白质和功能水平上都具有重要的数量意义，并服从于分子特征。这项研究提供了(I)该酶的Mr 64000底物结合亚基的氨基酸序列的测定，以及(Ii)组成功能完整泵的成分的计数和它们各自的作用。目的(I)深入了解这种新的易位酶的进化起源，并促进其在膜上的组织模型的建立。另一方面，目的(Ii)提供了对从序列数据得到的模型的独立测试，并有助于识别负责调节底物水解和H+-转位的结构元件。液泡膜PPase在植物细胞中的普遍存在，以及PPI作为高能磷酸盐极限情况的独特地位，将使所获得的结果具有广泛的生物能量意义。高等植物细胞的液泡膜(液泡膜)含有一种名为H+转运酶的酶。这种酶在高等植物中普遍存在，有助于跨膜电化学电位差的建立和维持。这种电化学电位差反过来对其他溶质的传输也是必要的。由于对这种重要的酶知之甚少，人们正在进行研究，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.