Reconstitution of Tonoplast H^+-ATPase from Mung Bean (Vigna radiata L.) Hypocotyls in Liposomes
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概要
- 論文の詳細を見る
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 immunoblotting 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-Mg^<2+>. ATP-Mg^<2+> 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, KNO_3. 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 KNO_3, 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.
- 日本植物生理学会の論文
著者
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Kasamo Kunihiro
National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries
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Saji Hikaru
National Institute For Environmental Studies
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Yamanishi Hiroyasu
National Food Research Institute
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Kagita Fumiko
National Food Research Institute
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Kagita Fumiko
Molecular Function Laboratory National Food Research Institute
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Kasamo K
National Institute Of Agrobiological Resoueces
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Yamanishi H
National Food Research Inst. Ibaraki Jpn
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Kasamo Kunihiro
National Food Res. Inst. Tsukuba
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