Identification and purification of substrate‐binding subunit of higher plant H⁺‐translocating inorganic pyrophosphatase

Abstract

The Hf-translocating inorganic pyrophosphatase (H+-PPasc) of Beta vacuolar membrane (tonoplast) vesicles has been purified by 90-fold from detergent-solubilized membranes and the MgPPi-binding subunit identified by affinity labeling. The purified enzyme has a specific activity of 1100 pmol/mg.h and contains one prevalent M, = 64000 polypeptide which strictly copurifies with activity.

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

“…The H+-PPase of Vigna tonoplast vesicles was purified by differential detergent solubilization (11) followed by anionexchange chromatography (4,11 (2) after precipitation of LPC, Triton X-100, and/or added phospholipid with ice-cold 5% (w/v) TCA, 2% (w/v) perchloric acid (20). To maximize the activity of the solubilized membranes and fractions from chromatography, the reaction media were supplemented with 1.33 mg/mL of sonicated type IV-S phospholipid (20).…”

“…The most homogeneous preparations of the tonoplast H+-PPase reported are those from storage root of B. vulgaris (4) and etiolated hypocotyls of V. radiata (11). The H+-PPase from Beta has been purified by 85-fold from Triton X-100-solubilized tonoplast vesicles by gel filtration on Sephacryl S400 and anion-exchange FPLC on Mono-Q to yield enzyme constituted of one major polypeptide of Mr 64,500 (4).…”

“…lOO, 1992 making a substantial contribution to the transtonoplast AAH+ (21), a major impediment to the structural analysis of the tonoplast H+-PPase is the confusion in the literature concerning its precise molecular identity. Britten et al (4) and Sarafian and Poole (24) attributed polypeptides of Mr 64,500 and 67,000, respectively, to the enzyme from red beet (Beta vulgaris) storage root, Maeshima and Yoshida (11) attributed a polypeptide of Mr 73,000 to the enzyme from etiolated hypocotyls of mung bean (Vigna radiata), and Chanson and Pilet (6) attributed polypeptides of Mr 37,000 to 45,000 to the enzyme from corn (Zea mays) roots. Thus, our objective in this investigation was to determine whether the reported differences in size and/or identity for the major subunits(s) of the H+-PPase from different sources have a structural basis or simply reflect differences in methodology between laboratories.…”

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

“…Protein purification [3][4][5][6], covalent modification [3,5], molecular cloning [7][8][9] and heterologous expression [9] have demonstrated the abundance, high catalytic activity, novelty and structural simplicity of the V-PPase, and patch clamp analyses implicate the pump in the primary translocation of both H ÷ and K ÷ into the vacuole [10]. However, despite these advances, very little is understood of how the one polypeptide species constituting the V-PPase accomplishes catalysis.…”

“…Potassium fluoride and Ca 2+, both of which are potent inhibitors of the V-PPase [18][19][20], inhibit medium P~-HOH oxygen exchange by more than 90% whereas addition of the type-specific V-ATPase "inhibitor bafilomycin A~ [21] causes 50% stimulation (Table 1). Likewise, pretreatment of vesicles with N-ethylmaleimide in the presence of both Mg 2+ and PPi [3], which irreversibly inhibits only 6% of the total PPi hydrolytic activity but 65% of the total ATP hydrolytic activity, diminishes the rate of exchange by only 27% when the F-ATPase inhibitor, NaN 3, is included in the reaction medium (Table 1). Consequently, any contribution from phosphohydro- lases other than the V-PPase is negligible.…”