AMINOMETHYLENEDIPHOSPHONATE - A POTENT TYPE-SPECIFIC INHIBITOR OF BOTH PLANT AND PHOTOTROPHIC BACTERIAL H-PYROPHOSPHATASES()

The suitability of different pyrophosphate (PPI) analogs as inhibitors of the vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) of tonoplast vesicles isolated from etiolated hypocotyls o f Vigna radiata was investigated. Five 1,1-diphosphonates and imidodip hosphate were tested for their effects on substrate hydrolysis by the V-PPase at a substrate concentration corresponding to the K-m of the e nzyme. The order of inhibitory potency (apparent inhibition constants, K-i(app) values, mu M, in parentheses) of the compounds examined was aminomethylenediphosphonate (1.8) > hydroxymethylenediphosphonate (5.7 ) congruent to ethane-1-hydroxy-1,l-diphosphonate (6.5)> imidodiphosph ate (12)> methylenediphosph on ate (68) > > dichloremethylenediphospho nate (>500). The specificity of three of these compounds, aminomethyle nediphosphonate, imidodiphosphate, and methylenediphosphonate, was det ermined by comparing their effects on the V-PPase and vacuolar H+-ATPa se from Vigna, plasma membrane H+-ATPase from Beta vulgaris, H+-PPi sy nthase 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-m of the V-PPase. Alth ough 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 cond itions. It is concluded that 1,1-diphosphonates, in general, and amino methylenediphosphonate, in particular, are potent type-specific inhibi tors of the V-PPase and its putative bacterial homolog, the H+-PPi syn thase of Rhodospirillum.