An existence of cold stable vacuolar H^+-ATPase in Plants and its structural properties.

绿豆（Vigna radiata L.）和豌豆（Pisum sativum L.）分别是冷敏感植物和耐寒植物的代表，液泡H^+- atp酶在体内的冷稳定性存在显著差异。绿豆幼苗冷暴露后，H^+- atp酶活性在冷处理开始后不久显著下降。相比之下，在豌豆幼苗中，H^+- atp酶在长时间的冷处理中保持高度活跃。在MgATP和C1^-、NO^- 2和NO^- 3阴离子存在的条件下，对离体液泡膜进行冷培养，液泡H^+- atp酶在冷诱导不稳定性方面也表现出显著差异，尤其是对阴离子浓度的敏感性。绿豆酶对阴离子的敏感性明显高于豌豆酶，特别是对NO^-_2的敏感性明显高于豌豆酶，且在低浓度条件下冷失活更为严重。mgatp依赖的H^+- atp酶的冷失活与外周区从膜上释放密切相关，这与来自广泛生物体的液泡型H^+- atp酶的释放方式相同。因此，酶复合物在体内和体外的冷稳定性被认为取决于酶的内在特性，这表明植物中存在“冷不稳定”和“冷稳定”类型。两种植物的H +- atp酶亚基组分的SDS-PAGE和免疫印迹分析显示，分子结构，特别是膜区表位（16kD），彼此不同，尽管其他次要亚基的分子质量也略有不同。本研究结果为研究植物液泡H +- atp酶冷稳定性的调控机制提供了新的思路。

The cold stability of vacuolar H^+-ATPase in vivo is markedly different between mung bean (Vigna radiata L.) and pea (Pisum sativum L.), the plants which are representatives of cold-sensitive and cold-tolerant species, respectively. Upon cold exposure of mung bean seedlings the H^+-ATPase activity declined significantly soon after the start of the cold treatment. In pea seedlings, by contrast, the H^+-ATPase remained highly active for long periods of the cold treatment. On cold incubation of isolated vacuolar membranes in vitro in the presence of MgATP and chaotropic anions such as C1^-, NO^-_2 and NO^-_3, the vacuolar H^+-ATPases exhibited also a marked difference in the cold-induced destabilization between these plants, especially in the sensitivity to the concentration of the anions. The enzyme from mung bean appeared to be more susceptible to the anions, particularly to NO^-_2, than the enzyme from pea, resulting in more severe cold-inactivation at the lower concentrations. The MgATP-dependent cold inactivation of the H^+-ATPase was closely associated with a release of the peripheral sectors from the membranes in the same way as in vacuolar-type H^+-ATPases from wide range of organisms. Thus, the cold stability of the enzyme complex both in vivo and in vitro is considered to be dependent on the intrinsic properties of the enzyme, suggesting the existence of "cold-labile" and "cold-stable" types in plants. SDS-PAGE and immunoblot analysis of the subunit components of the H^+-ATPase from both plants revealed that the molecular structures, especially the epitopes of the membrane sectors (16kD), differ each other, albeit that the molecular masses of other minor subunits are also slightly different. These findings in the present study are considered to provide a new insight into the mechanism to control the cold stability of vacuolar H^+-ATPase in plants.