Effects of Cerebroside and Cholesterol on the Reconstitution of Tonoplast H^+-ATPase Punfied from Mung Bean(Vigna radiata L.)Hypocotyls in Liposomes : MEMBRANES AND BIOENERGETICS

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概要

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• For an examination of the effects of cholesterol and cerebroside on the rate and extent of proton-pumping across the membranes of proteoliposomes prepared with tonoplast H^+-ATPase, the tonoplast H^+-ATPase of mung bean (Vigna radiata L.) was purified by fast protein liquid chromatography (FPLC) and incorporated into liposomes prepared from asolectin and cholesterol or cerebroside. Proteoliposomes were formed after the removal of Triton X-100 from a mixture of Triton X-100, asolectin and purified tonoplast H^+-ATPase by passage through an Ampure DT column. Proteoliposomes prepared from cholesterol and asolectin at a ratio of 45 : 55 (w/w) and at a ratio of lipid to protein of 200 : 1 (w/w) gave the largest pH gradient, as determined by the ATP-generated quenching of quinacrine fluorescence. In the presence of cholesterol, the pH gradient formed across the membranes of proteoliposomes and the average diameter of proteoliposomes increased about two-fold. The initial rate of proton-pumping decreased to 20% of that observed with proteoliposomes prepared from asolectin alone. The addition of cerebroside to asolectin at a ratio of 5 : 95 (w/w) caused a 1.6-fold increase in the maximum pH gradient with-out any significant change in the initial rate of proton-pumping or the diameter of proteoliposomes, but the maximum pH gradient decreased greatly at ratios above 20 : 80 (w/w). The maximum pH gradient was transient and decreased spontaneously when only asolectin was used to prepare proteoliposomes, or when cerebroside and asolectin were used together. Disappearance of the proton gradient once it had formed and/or leakage of protons were suppressed by cholesterol at ratios above 30 : 70 (w/w). It was clear, therefore, that cholesterol and asolectin at ratios 30 : 70 (w/w) to 45 : 55 (w/w) formed larger and more stable proteoliposomes than did asolectin alone.

• 日本植物生理学会の論文

著者

• Kasamo Kunihiro

  Molecular Function Laboratory Biological Function Division National Food Research Institute

• YAMANISHI Hiroyuki

  Molecular Function Laboratory, Biological Function Division, National Food Research Institute

• Yamanishi Hiroyuki

  Molecular Function Laboratory Biological Function Division National Food Research Institute:(present

関連論文

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• Changes in the Cytoplasmic and Vacuolar pH in Intact Cells of Mung Bean Root-Tips under High-NaCl Stress at Different External Concentrations of Ca^ Ions : ENVIRONMENTAL AND STRESS RESPONSES
• Stimulation of the Extrusion of Protons and H^+-ATPase Activities with the Decline in Pyrophosphatase Activity of the Tonoplast in Intact Mung Bean Roots under High-NaCl Stress and Its Relation to External Levels of Ca^ Ions
• Modulation of the Activity of Purified Tonoplast H^+-ATPase from Mung Bean (Vigna radiata L.) Hypocotyls by Various Lipids
• Low Temperature-Induced Changes in the Thermotropic Properties and Fatty Acid Composition of the Plasma Membrane and Tonoplast of Cultured Rice (Oryza sativa L.) Cells : ENVIRONMENTAL AND STRESS RESPONSES : MEMBRANES AND BIOENERGETICS
• Functional Reconstitution of Plasma Membrane H^+-ATPase from Mung Bean ( Vigna radiata L.) Hypocotyls in Liposomes Prepared with Various Molecular Species of Phospholipids
• Effects of Cerebroside and Cholesterol on the Reconstitution of Tonoplast H^+-ATPase Punfied from Mung Bean(Vigna radiata L.)Hypocotyls in Liposomes : MEMBRANES AND BIOENERGETICS

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