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

植物冷稳定液泡H^-ATP酶的存在及其结构特性。

基本信息

批准号：
05454012
负责人：
YOSHIDA Shizuo
金额：
$ 4.29万
依托单位：
The Institute of Low Temperature Science Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

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.

绿豆（Vigna radiata L.）和豌豆（Pisum sativum L.），分别是冷敏感和耐寒植物的代表。绿豆幼苗经冷处理后，H^+-ATPase活性在冷处理开始后不久即显著下降。相比之下，豌豆幼苗的H^+-ATP酶在长时间的低温处理中仍保持高度活性。离体液泡膜在MgATP和离液阴离子（如Cl ^-、NO^-2和NO^-3）存在下冷孵育时，液泡H^+-ATP酶在冷诱导的不稳定性方面也表现出明显的差异，特别是对阴离子浓度的敏感性。绿豆酶比豌豆酶更易受阴离子的影响，尤其是对NO2-的影响，在较低浓度下，绿豆酶的冷失活更为严重。MgATP依赖的H^+-ATP酶冷失活与细胞膜周边部分的释放密切相关，其方式与许多生物体中的液泡型H^+-ATP酶相同。因此，在体内和体外的酶复合物的冷稳定性被认为是依赖于酶的内在性质，这表明在植物中存在的“冷不稳定”和“冷稳定”类型。对两种植物H^+-ATPase亚基组分的SDS-PAGE和免疫印迹分析表明，分子结构，特别是膜部分（16 kD）的表位，彼此不同，尽管其他次要亚基的分子量也略有不同。这些结果为植物液泡H^+-ATPase冷稳定性的调控机制提供了新的认识。