Department of Botany, University of Maryland, College Park, Maryland 20742.
Plant Physiol. 1992 May;99(1):170-9. doi: 10.1104/pp.99.1.170.
The vacuolar H(+)-translocating ATPase (H(+)-ATPase), originally reported to consist of three major subunits, has been further purified from oat roots (Avena sativa var Lang) to determine the complete subunit composition. Triton-solubilized ATPase activity was purified by gel filtration on Sephacryl S400 and ion-exchange chromatography (Q-Sepharose). ATP hydrolysis activity of purified preparations was inhibited by 100 nanomolar bafilomycin A(1), a specific vacuolar-type ATPase inhibitor. The purified oat H(+)-ATPase (relative molecular weight = 650,000) was composed of polypeptides of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kilodaltons. To analyze the organization of the H(+)-ATPase subunits, native vacuolar membranes were treated with KI and MgATP to dissociate peripheral proteins. Release of 70, 60, 44, 42, 36, and 29 kilodalton polypeptides from the membrane was accompanied by a loss of ATP hydrolysis and ATP-dependent H(+)-pumping activities. Five of the peripheral subunits were released from the membrane as a large complex of 540 kilodaltons. Vesicles that had lost the peripheral sector of the ATPase could hold a pH gradient generated by the proton-translocating pyrophosphatase, suggesting that the integral sector of the ATPase did not form a H(+)-conducting pathway. Negative staining of native vesicles revealed knob-like structures of 10 to 12 nanometers in dense patches on the surface of vacuolar membranes. These structures were removed by MgATP and KI, which suggested that they were the peripheral sectors of the H(+)-ATPase. These results demonstrate that the vacuolar H(+)-ATPase from oat roots has 10 different subunits. The oat vacuolar ATPase is organized as a large peripheral sector and an integral sector with a subunit composition similar, although not identical to, other eukaryotic vacuolar ATPases. Variations in subunit composition observed among several ATPases support the idea that distinct types of vacuolar H(+)-ATPases exist in plants.
液泡 H(+)-转运 ATP 酶（H(+)-ATPase）最初被报道由三个主要亚基组成，后来从燕麦根（Avena sativa var Lang）中进一步纯化以确定完整的亚基组成。用 Triton 溶解的 ATP 酶活性通过 Sephacryl S400 和离子交换层析（Q-Sepharose）进行凝胶过滤纯化。纯化制剂的 ATP 水解活性被 100 纳摩尔巴弗洛霉素 A(1)抑制，巴弗洛霉素 A(1)是一种特异性的液泡型 ATP 酶抑制剂。纯化的燕麦 H(+)-ATP 酶（相对分子质量=650,000）由 70、60、44、42、36、32、29、16、13 和 12 千道尔顿的多肽组成。为了分析 H(+)-ATP 酶亚基的组成，用 KI 和 MgATP 处理天然液泡膜以解离外周蛋白。膜释放 70、60、44、42、36 和 29 千道尔顿多肽伴随着 ATP 水解和 ATP 依赖的 H(+)泵出活性的丧失。五个外周亚基从膜上释放出来形成一个 540 千道尔顿的大复合物。失去 ATP 酶外周部分的囊泡可以保持质子转运焦磷酸酶产生的 pH 梯度，表明 ATP 酶的整合部分没有形成 H(+)传导途径。天然囊泡的负染色显示在液泡膜表面的致密斑块上有 10 到 12 纳米的结节样结构。这些结构被 MgATP 和 KI 去除，这表明它们是 H(+)-ATP 酶的外周部分。这些结果表明，来自燕麦根的液泡 H(+)-ATP 酶有 10 个不同的亚基。燕麦液泡 ATP 酶的结构组织为一个大的外周部分和一个整合部分，其亚基组成与其他真核液泡 ATP 酶相似，尽管不完全相同。几种 ATP 酶中观察到的亚基组成的变化支持这样一种观点，即在植物中存在不同类型的液泡 H(+)-ATP 酶。
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