l h e suitability of different pyrophosphate (PPi) analogs as inhibitors of the vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1 .l) of tonoplast vesicles isolated from etiolated hypocotyls of Vigna radiata was investigated. Five 1,l-diphosphonates and imidodiphosphate were tested for their effects on substrate hydrolysis by the V-PPase at a substrate concentration corresponding to the K,,, of the enzyme. l h e order of inhibitory potency (apparent inhibition constants, KiaPP values, p~, in parentheses) of the compounds examined was aminomethylenediphosphonate (1.8) > hydroxymethylenediphosphonate (5.7) I ethane-l-hydroxy-1,l-diphosphonate (6.5) > imidodiphosphate (12) > methylenediphosphonate (68) >> dichloromethylenediphosphonate (>500). l h e specificity of three of these compounds, aminomethylenediphosphonate, imidodiphosphate, and methylenediphosphonate, was determined by comparing their effects on the V-PPase and vacuolar H+-ATPase from Vigna, plasma membrane H+-ATPase from Beta vulgaris, H+-PPi synthase of chromatophores prepared from Rhodospirillum rubrum, soluble PPase from Saccharomyces cerevisiae, alkaline phosphatase from bovine intestinal mucosa, and nonspecific monophosphoesterase from Vigna at a PPi concentration equivalent to 10 times the K,,, of the V-PPase. Although all three PPi analogs inhibited the plant V-PPase and bacterial H+-PPi synthase with qualitatively similar kinetics, whether substrate hydrolysis or PPi-dependent H+-translocation was measured, neither the vacuolar H+-ATPase nor plasma membrane H+-ATPase nor any of the non-V-PPase-related PPi hydrolases were markedly inhibited under these conditions. It is concluded that 1,l-diphosphonates, in general, and aminomethylenediphosphonate, in particular, are potent type-specific inhibitors of the V-PPase and its putative bacterial homolog, the H+-PPi synthase of Rhodospirillum.It is now established that PPi is a major energy source for electrogenic H+ translocation across the vacuolar membrane of plant cells (Rea et al., 1992b;Rea and Poole, 1993) * Corresponding author; fax 1-215-898-8780. 153transtonoplast H+ electrochemical potential difference (Rea et al., 1992b;Rea and Poole, 1993).Interest in the V-PPase derives not only from its exclusive use of PPi as energy source but also from its apparently unique evolutionary status among the various categories of primary ion translocases and PPi hydrolases. Data base searches of the deduced sequences of the polypeptides encoded by cDNAs corresponding to the major substrate-binding subunit of the V-PPase reveal no detectable homology between this pump and other sequenced ion translocases (Rea et al., 1992b;Sarafian et al., 1992). Sidarly, close phylogenic links between the V-PPase and the soluble, nonenergy-conserving PPases of both prokaryotes and eukaryotes are unlikely. A11 characterized soluble PPases have different subunit sizes from the V-PPase (Coopennan et al., 1992), and none of the known sequences for soluble PPases align with the deduced sequence of the V-PPa...

The results of analyses of the steady-state kinetics of the vacuolar H+-translocating pyrophosphatase (V-PPase) of native tonoplast vesicles isolated from etiolated hypocotyls of fignu rudiutu (mung bean) and purified enzyme from the same source under a wide range of Mg", pyrophosphate (PPJ and K' concentrations are consistent with a minimal reaction scheme in which dimagnesium pyrophosphate is the active substrate species and catalysis is mediated by preformed enzyme-Mg'+ complex. When account is taken of the sensitivity of the V-PPase to ionic strength, additional kinetic interactions are not required to describe the behavior of the enzyme. N-Ethylmaleimide-protection assays show that the dissociation constant for Mg" binding in the absence of PP, is an order of magnitude smaller than that estimated from the steady-state kinetics of PP, hydrolysis. Two distinct Mg"-binding sites are therefore invoked. Since the protective action of Mg2+ is independent of the nature of the monovalent cation and Mg" and K' do not compete during substrate hydrolysis, divalent and monovalent cations are concluded to bind at separate sites. The pH dependencies of the kinetic parameters are consistent with the participation of groups of pK, 5.7 and 8.6 in substrate binding and groups of pK, 6.1 and 9.0 in the substrate-conversion step, indicating that at least four ionizable groups are essential for catalysis. These findings are discussed with respect to the reaction mechanism of the V-PPase and the potential regulatory significance of cytosolic free Mg2+ and K+ in vivo.Plant cells contain alternative metabolic pathways which utilize nucleotides or inorganic pyrophosphate (PP,) as energy sources. While the full significance of this phenomenon remains to be determined, this pattern of alternate proximate energy sources is exemplified by the presence of two parallel H' pumps in the vacuolar membrane (tonoplast). These are the vacuolar H'-ATPase (V-ATPase), an enzyme common to the endomembranes of all characterized eukaryotes (Nelson, 1992;Sze et al., 1992), and a vacuolar H i -translocating inorganic pyrophosphatase (V-PPase), which is ubiquitous in plants but otherwise known in only a few phototrophic bacteria (Baltscheffsky, 1978;Rea et al., 1992a;Rea and Poole, 1993). 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 constitu...

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l,l-Diphosphonate analogs of pyrophosphate, containmg an amino or a hydroxyl group on the bridge carbon atom, are potent mhibitors of the H*-translocating pyrophosphatases of chromatophores prepared from the bacterium R~udospzr~~Zut~l rubnon and vacuolar membraI~e vesicles prepared from the plant yigna vu&&z. The mhibttton constant for aminomethylenediphosphonate, which bmds competitively with respect to substrate, ts below 2 PM. Rat liver mitochondrial pyrophosphatase is two orders of magnitude less sensitive to this compound but extremely sensitive to imidodiphosphate. By contrast, fluoride is highly effective only against the mitochondrial pyrophosphatase. It 1s concluded that the mttochondrial pyrophosphatase and the H+-pyrophosphatases of ehromatophores and vacuolar membranes belong to two different classes of enzyme.

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