The Institute of Low Temperature Science, Hokkaido University, Sapporo 060, Japan.
Plant Physiol. 1986 Jan;80(1):152-60. doi: 10.1104/pp.80.1.152.
Plasma membrane was isolated in a uniform population and with a high purity from chilling-sensitive etiolated young seedlings of Vigna radiata (mung bean) utilizing an aqueous two polymer phase separation system and subsequent sucrose density gradient. The isolated plasma membrane was associated with vanadate-sensitive and KNO(3)-insensitive ATPase. The ATPase has high specificities both for substrate and Mg(2+) ion with optimum pH at 6.5. It was slightly stimulated by monovalent anions, especially Cl(-). Proton ionophores such as gramicidin D and carbonyl cyanide p-trifluoromethoxyphenylhydrazone did not stimulate the enzyme activity. The ATPase is apparently latent and highly stimulated by the addition of detergents such as Triton X-100. A maximum stimulation was achieved by the addition of 0.02% Triton X-100. After treatment with proteinase K in an isotonic buffer solution, the enzyme activity was less affected, whereas the peptides were specifically digested. Based on these facts, the isolated plasma membrane vesicles appear to be tightly sealed and in a right-side-out orientation. The plasma membrane ATPase had two inflection points at higher (18.9 degrees C) and lower (6.7 degrees C) temperatures on the Arrhenius plots of the activity. The lower inflection temperature apparently coincided with that of the anisotropy parameter of embedded 1,6-diphenyl-1,3,5-hexatriene, indicating that the membrane bound ATPase activity was affected by a phase transition of membrane lipids and/or temperature-dependent conformational changes in the enzyme molecules per se. Considering the fact that the plant material used here is highly sensitive to chilling temperatures and injured severely by exposure to temperatures below 5 degrees C for a relatively short period, the thermotropic properties of membrane molecules are considered to be involved in the mechanism of chilling injury.
从冷敏感白化幼苗的绿豆（绿豆）利用水两聚合物相分离系统和随后的蔗糖密度梯度，以均一的群体和高纯度分离质膜。分离的质膜与钒酸盐敏感和 KNO （3） - 不敏感的 ATPase 有关。ATPase 对底物和 Mg （2 +）离子具有很高的特异性，最佳 pH 值为 6.5。它受单价阴离子的轻微刺激，特别是 Cl （-）。质子载体如短杆菌肽 D 和羰基氰化物 p-三氟甲氧基苯腙不刺激酶活性。ATPase 显然是潜伏的，并且高度受去污剂如 Triton X-100 的添加的刺激。通过添加 0.02％Triton X-100 可实现最大刺激。在等渗缓冲溶液中用蛋白酶 K 处理后，酶活性受影响较小，而肽被特异性消化。基于这些事实，分离的质膜囊泡似乎被紧密密封并处于正确的向外取向。质膜 ATPase 在活性的 Arrhenius 图上在较高（18.9°C）和较低（6.7°C）温度下具有两个拐点。较低的拐点温度显然与嵌入 1,6-二苯基-1,3,5-己三烯的各向异性参数一致，表明膜结合 ATPase 活性受膜脂的相变和/或本身温度依赖性的酶分子构象变化的影响。考虑到这里使用的植物材料对冷温度非常敏感并且在相对较短的时间内暴露于 5°C 以下的温度下会严重受损的事实，膜分子的热性质被认为参与了冷害的机制。
