Department of Biology, McGill University, Montreal, Quebec, CANADA H3A 1B1.
Plant Physiol. 1986 May;81(1):126-9. doi: 10.1104/pp.81.1.126.
Membrane vesicles derived from the tonoplast of Beta vulgaris L. possess two predominant phosphohydrolase activities: a Cl(-)-stimulated, NO(3) (-)-inhibited ATPase, and a K(+)-stimulated, Na(+)-inhibited inorganic pyrophosphatase (PPase). The solubilization of tonoplast vesicles with 2% (w/v) Triton X-100 containing 4 millimolar MgCl(2) and 1 millimolar ethylenediamine tetracetic acid, as protectants, gives high yields of both the ATPase and PPase in soluble form. Chromatography of the solubilized membranes on Sephacryl-400 results in the separation of the two enzymes. The PPase and ATPase are purified 4- and 17-fold, respectively, with quantitative recovery. The separated enzymes show negligible activity towards the other's substrate and the separated PPase only hydrolyzes pyrophosphate. The separated enzymes show mineral ion requirements identical to those of the corresponding pump and hydrolytic activities in native tonoplast and both solubilized enzymes are subject to phospholipid activation.
一种是 Cl(-) 刺激、NO(3) (-) 抑制的 ATP 酶，另一种是 K(+) 刺激、Na(+) 抑制的无机焦磷酸酶（PPase）。用含 4 毫摩尔氯化镁和 1 毫摩尔乙二胺四乙酸的 2%（重量/体积）Triton X-100 溶解类囊体小泡，并作为保护剂，可以得到高产量的可溶性 ATP 酶和 PPase。将溶解的膜在 Sephacryl-400 上进行层析，可以将两种酶分离。PPase 和 ATPase 的纯化倍数分别为 4 倍和 17 倍，回收率定量。分离的酶对对方的底物几乎没有活性，分离的 PPase 仅水解焦磷酸。分离的酶显示出与天然类囊体中相应泵和水解活性相同的矿物离子需求，并且两种可溶性酶都受磷脂激活的影响。
