Reconstitution of Tonoplast H+-ATPase from Mung Bean (Vigna radiata L.) Hypocotyls in Liposomes

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Abstract
Reconstituted proteoliposomes of tonoplast ATPase are formed on solubilization of tonoplast membranes from mung bean (Vigna radiata L.) with deoxycholate (DOC) in the presence of a mixture of soybean phospholipids (asolectin), after removal of DOC by passage through a PD-10 column (Pharmacia). This method is ideal because of its simplicity and rapidity. Selective insertion of sets of tonoplast H+-ATPase polypeptides (68 kDa, 60 kDa, 16 kDa and several minor polypeptides) into liposomes using this method was confirmed by SDS-PAGE and immuno-blotting with antibodies raised against 68-kDa and 60-kDa polypeptides. Pumping of protons across the membranes of the proteoliposomes was demonstrated by quinacrine-fluorescence quenching in the presence of ATP-Mg2+. ATP-Mg2+ was shown to be the preferred substrate in both reconstituted and native tonoplast vesicles, and its optimum concentration was 0.75 to 3.0 mM. Quenching was completely abolished by a channel-forming ionophore, gramicidin D, and an inhibitor of tonoplast H+-ATPase, KNO3. Antibodies to 68-kDa and 60-kDa peptides partially inhibited the pumping of protons. The rate of pumping of protons increased with the number of proteoliposomes, the maximal concentration of which was equivalent to 250 μg of protein per reaction mixture. The optimum pH for pumping was 6.5 when inside of proteoliposomes were loaded pH at 7.2. The rate of pumping of protons was reduced when proteoliposomes were made using asolectin and cholesterol at 3 : 1 (w/w), as compared with those made with asolectin alone. The ATPase activity in reconstituted proteoliposomes was inhibited by KNO3, with half-maximal inhibition at approximately 7 mM. The enzyme actively hydrolyzed ATP in preference to GTP, CTP, UTP, and ADP, but it did not hydrolyze pNPP or AMP. Antibodies against the 60-kDa polypeptide strongly inhibited ATPase activity as compared to antibodies against the 68-kDa polypeptide. The results obtained in this study demonstrate directly that functional tonoplast H+-ATPase can be inserted selectively into liposomes.

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