Aminomethylenediphosphonate: A Potent Type-Specific Inhibitor of Both Plant and Phototrophic Bacterial H+-Pyrophosphatases

Abstract

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...

Heterologous expression of plant vacuolar pyrophosphatase in yeast demonstrates sufficiency of the substrate-binding subunit for proton transport.

“…4A), whereas membranes prepared from untransformed yeast (data not shown) and glucose-grown transformants show negligible PPi-dependent H+ translocation. H+ pumping by the heterologously expressed V-PPase has a near-absolute K+ requirement and is strongly inhibited by aminomethylenediphosphonate, as is the enzyme of plant vacuolar membrane vesicles (4,19). Provision of 50 mM KCl elicits high rates of intravesicular acidification, and substitution of this salt with the same concentration of choline chloride or inclusion of 20 ,uM aminomethylenediphosphonate in the fluorescence assay medium markedly diminishes the initial rate and extent of intravesicular acidification (Fig.…”

Characterization of a Vacuolar Pyrophosphatase in Trypanosoma brucei and Its Localization to Acidocalcisomes

“…2D). The effective concentrations of KF, AMDP, and IDP were similar to those that inhibit plant V-H ϩ -PPase activity (47,50). The effects of different known H ϩ -ATPase inhibitors on PP i -dependent acridine orange uptake by permeabilized trypomastigotes were also investigated.…”

Human Platelet Dense Granules Contain Polyphosphate and Are Similar to Acidocalcisomes of Bacteria and Unicellular Eukaryotes

“…As it occurs with acidocalcisomes of T. brucei (46), pyrophosphatase activity was enriched in the dense granule fraction of human platelets and this activity was inhibited by AMDP, an inhibitor of V-H ϩ -PPases (Fig. 9A) (37). Although one study reported PP i -induced acidification of trans-cisternal elements of rat liver Golgi membranes (47), there have been no reports on the presence of genes with similarity to those encoding V-H ϩ -PPases of bacteria, plants, and protists.…”