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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.)分别是冷敏感植物和耐冷植物的代表，体内液泡H^+-ATP酶的冷稳定性存在显着差异。绿豆幼苗在冷暴露后，H^+-ATP酶活性在冷处理开始后不久就显着下降。相比之下，在豌豆幼苗中，H^+-ATP酶在长时间的冷处理中仍保持高度活性。在存在 MgATP 和离液阴离子（如 C1^-、NO^-_2 和 NO^-_3）的情况下体外冷孵育分离的液泡膜时，液泡 H^+-ATP 酶在这些植物之间的冷诱导不稳定方面也表现出显着差异，特别是对阴离子浓度的敏感性。来自绿豆的酶似乎比来自豌豆的酶更容易受到阴离子，特别是NO^-_2的影响，导致在较低浓度下更严重的冷失活。 H^+-ATP酶的MgATP依赖性冷失活与膜周围部分的释放密切相关，其方式与来自多种生物体的液泡型H^+-ATP酶相同。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，表明植物中存在“冷不稳定”和“冷稳定”类型。对两种植物的 H^+-ATPase 亚基成分进行 SDS-PAGE 和免疫印迹分析表明，尽管其他次要亚基的分子量也略有不同，但分子结构，特别是膜部分（16kD）的表位彼此不同。本研究的这些发现被认为为控制植物液泡H^+-ATP酶的冷稳定性机制提供了新的见解。