Common Identity of Substrate Binding Subunit of Vacuolar H⁺-Translocating Inorganic Pyrophosphatase of Higher Plant Cells

Abstract

There have been conflicting reports in the literature concerning the polypeptide composition of the vacuolar H+-translocating inorganic pyrophosphatase (tonoplast H+-PPase) of plant cells. The major subunit(s) of the enzyme have been attributed to polypeptides of relative molecular weight (Mr) 64,500 (Beta vulgaris), 67,000 (Beta vulgaris), 73,000 (Vigna radiata), and 37,000 to 45,000 (Zea mays). Here, we reconcile these differences to show, through the combined application of independent purification, affinitylabeling, sequencing, and immunological procedures, that the major polypeptide associated with the H+-PPase from all of these organisms, and Arabidopsis thaliana, corresponds to the same moiety. The principal polypeptide components of the H+-PPase purified from Beta and Vigna by independent procedures have similar apparent subunit masses when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under

Steady‐state kinetics of substrate hydrolysis by vacuolar H+‐pyrophosphatase

“…The major (Mg" + PPJ-binding (catalytic) subunit has been identified (Britten et al, 1989;Rea et al, 1992b) and purified (Britten et al, 1989;Maeshima and Yoshida, 1989;Sarafian and Poole, 1989; a]., 1992b); partially purified preparations of the enzyme have been reconstituted to yield proteoliposomes active in PP,-dependent, electrogenic H' translocation (Britten et al, 1992); cDNAs encoding the major subunit and its isoforms have been isolated and sequenced (Sarafian et al, 1992;Rea et al 1993;Tanaka et al, 1993). Despite these recent developments, there is, however, still a paucity of reliable information concerning the identity of the catalytic and regulatory ligands which interact with the V-PPase.…”

“…The major (Mg" + PPJ-binding (catalytic) subunit has been identified (Britten et al, 1989;Rea et al, 1992b) and purified (Britten et al…”

“…Both enzymes catalyze electrogenic H'-translocation from the cytosol to vacuole lumen to establish an inside-acid pH difference (dpH) and inside-positive electrical potential difference ( d w ) which is employed to energize the H+-coupled, secondary transport of solutes across the vacuolar membrane (Sze, 1985 ;Blumwald, 1987 The potential bioenergetic impact of the V-PPase is indicated by its intrinsic catalytic activity and abundance. The enzyme is capable of generating a steady-state transtonoplast H+-electrochemical potential difference (dPH +) of similar, or greater, magnitude than the V-ATPase on the same membrane (Johannes and Felle, 1989;Maeshima and Yoshida, 1989; Pope and Leigh, 1988;Rea et al, 1992b) and abundance estimates indicate the the V-PPase constitutes between 1 % (Beta vulgaris storage root; Britten et al, 1989 ;Rea et al, 1992b) and 5-10% of total vacuolar membrane protein (Vigna radiatu hypocotyls; Maeshima and Yoshida, 1989 ;Rea et al, 1992b). When account is taken of the fact that the vacuole of a typical mature plant cell represents 50-90% of total intracellular volume (Raven, 1987) and hydrolysis by the V-PPase may be the sole mechanism for disposal of cytosolic PP, (Weiner et al, 1987;Takeshige and Tazawa, 1989), it is clear that the V-PPase has the capacity to substantially influence overall cellular energy status.…”

Oxygen exchange reactions catalyzed by vacuolar H+‐translocating pyrophosphatase Evidence for reversible formation of enzyme‐bound pyrophosphate

“…2). In conjunction with the inhibitor sensitivities of the reaction, these results clearly show that the V-PPase, the dominant phosphohydrolase activity associated with vacuolar membrane vesicles prepared from V. radiata [4,14], is responsible for the exchange reaction. First, medium Pi-HOH exchange exhibits a second-order dependence on Pi concentration (Fig.…”

“…Vacuolar membrane vesicles were purified from etiolated hypocotyls of 14. radiata by differential and density gradient centrifugation [14], suspended in suspension medium (10% (w/v) glycerol, 1 mM ethylene glycol bis(b-aminoethyl ether)N,N'-tetraacetate (EGTA), 1 mM dithiothreitol, 5 mM Tris-MES, pH 7.2), frozen in liquid N 2 and stored at -85°C. Purified enzyme was prepared by gel filtration as described previously [11,15].…”