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.
