Parry R V, Turner J C, Rea P A
Department of Biochemistry, Institute of Arable Crops Research, Harpenden, Herts, United Kingdom.
J Biol Chem. 1989 Nov 25;264(33):20025-32.
A fast protein liquid chromatography procedure for purification of the V-type H+-ATPase from higher plant vacuolar membrane to yield near-homogeneous enzyme with a specific activity of 20-25 mumol/mg.min is described. When precautions are taken to ensure the quantitative recovery of protein before sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the preparation is found to be constituted of seven major polypeptides of 100, 67, 55, 52, 44, 32, and 16 kDa, respectively, and two minor components of 42 and 29 kDa. The 52-, 44-, and 32-kDa polypeptides do not cross-react with antisera raised to the 67- and 55-kDa subunits of the enzyme, and two independent sample preparation procedures yield the same apparent subunit composition. The additional polypeptides are not breakdown products or aggregates of the previously identified subunits of the ATPase. The ATPase of tonoplast vesicles is subject to MgATP-dependent cold inactivation, and the conditions for inactivation are identical to those for the bovine chromaffin granule H+-ATPase (Moriyama, Y., and Nelson, N. (1989) J. Biol. Chem. 264, 3577-3582). Cold inactivation is accompanied by the detachment of five major polypeptides of 67, 55, 52, 44, and 32 kDa from the membrane, and all five components co-migrate with the corresponding polypeptides of the purified ATPase upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 100- and 16-kDa polypeptides of the ATPase are not removed from the membrane during cold inactivation, but the latter can be purified to homogeneity by chloroform:methanol extraction of the fast protein liquid chromatography-purified enzyme. It is concluded that the tonoplast H+-ATPase is constituted of 6-7 major polypeptides organized into a peripheral sector comprising the 67-, 55-, 52-, 44-, and 32-kDa components and an integral sector consisting of the 100- and 16-kDa polypeptides. The V-type H+-ATPase from animal endomembranes and higher plant vacuolar membranes therefore have remarkably similar subunit compositions and gross topographies.
本文描述了一种快速蛋白质液相色谱法，用于从高等植物液泡膜中纯化V型H⁺-ATP酶，以获得比活性为20 - 25 μmol/mg·min的近均一酶。在采取预防措施确保十二烷基硫酸钠-聚丙烯酰胺凝胶电泳前蛋白质定量回收时，发现该制剂分别由100、67、55、52、44、32和16 kDa的七种主要多肽以及42和29 kDa的两种次要成分组成。52、44和32 kDa的多肽不与针对该酶67和55 kDa亚基产生的抗血清发生交叉反应，并且两种独立的样品制备方法产生相同的表观亚基组成。额外的多肽不是ATP酶先前鉴定的亚基的降解产物或聚集体。液泡膜囊泡的ATP酶会受到MgATP依赖的冷失活作用，失活条件与牛嗜铬颗粒H⁺-ATP酶相同（森山洋和尼尔森，N.（1989年）《生物化学杂志》264，3577 - 3582）。冷失活伴随着67、55、52、44和32 kDa的五种主要多肽从膜上脱离，并且在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳时，所有这五种成分与纯化的ATP酶的相应多肽共迁移。ATP酶的100和16 kDa多肽在冷失活过程中不会从膜上被去除，但后者可以通过对快速蛋白质液相色谱纯化的酶进行氯仿：甲醇提取来纯化至均一。得出的结论是，液泡膜H⁺-ATP酶由6 - 7种主要多肽组成。这些多肽组织成一个外周部分，包括67、55、52、44和32 kDa的成分，以及一个由100和16 kDa多肽组成的整合部分。因此，来自动物内膜和高等植物液泡膜的V型H⁺-ATP酶具有非常相似的亚基组成和总体拓扑结构。
