Salvucci M E, Osteryoung K W, Crafts-Brandner S J, Vierling E
Western Cotton Research Laboratory, United States Department of Agriculture-Agricultural Research Service, 4135 East Broadway Road, Phoenix, AZ 85040-8803, USA.
Plant Physiol. 2001 Nov;127(3):1053-64.
Heat stress inhibits photosynthesis by reducing the activation of Rubisco by Rubisco activase. To determine if loss of activase function is caused by protein denaturation, the thermal stability of activase was examined in vitro and in vivo and compared with the stabilities of two other soluble chloroplast proteins. Isolated activase exhibited a temperature optimum for ATP hydrolysis of 44 degrees C compared with > or =60 degrees C for carboxylation by Rubisco. Light scattering showed that unfolding/aggregation occurred at 45 degrees C and 37 degrees C for activase in the presence and absence of ATPgammaS, respectively, and at 65 degrees C for Rubisco. Addition of chemically denatured rhodanese to heat-treated activase trapped partially folded activase in an insoluble complex at treatment temperatures that were similar to those that caused increased light scattering and loss of activity. To examine thermal stability in vivo, heat-treated tobacco (Nicotiana rustica cv Pulmila) protoplasts and chloroplasts were lysed with detergent in the presence of rhodanese and the amount of target protein that aggregated was determined by immunoblotting. The results of these experiments showed that thermal denaturation of activase in vivo occurred at temperatures similar to those that denatured isolated activase and far below those required to denature Rubisco or phosphoribulokinase. Edman degradation analysis of aggregated proteins from tobacco and pea (Pisum sativum cv "Little Marvel") chloroplasts showed that activase was the major protein that denatured in response to heat stress. Thus, loss of activase activity during heat stress is caused by an exceptional sensitivity of the protein to thermal denaturation and is responsible, in part, for deactivation of Rubisco.
热胁迫通过降低核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco）激活酶对Rubisco的激活作用来抑制光合作用。为了确定激活酶功能丧失是否由蛋白质变性引起，我们在体外和体内检测了激活酶的热稳定性，并将其与另外两种可溶性叶绿体蛋白的稳定性进行了比较。分离得到的激活酶对ATP水解的最适温度为44℃，而Rubisco羧化反应的最适温度≥60℃。光散射表明，在有和没有ATPγS存在的情况下，激活酶分别在45℃和37℃发生解折叠/聚集，而Rubisco在65℃发生解折叠/聚集。将化学变性的硫氰酸酶添加到热处理的激活酶中，在与导致光散射增加和活性丧失的温度相似的处理温度下，将部分折叠的激活酶捕获在不溶性复合物中。为了检测体内热稳定性，在硫氰酸酶存在的情况下，用去污剂裂解热处理的烟草（黄花烟草品种Pulmila）原生质体和叶绿体，并通过免疫印迹法测定聚集的靶蛋白量。这些实验结果表明，激活酶在体内的热变性发生温度与分离的激活酶变性温度相似，且远低于使Rubisco或磷酸核酮糖激酶变性所需的温度。对来自烟草和豌豆（豌豆品种“小奇迹”）叶绿体的聚集蛋白进行的埃德曼降解分析表明，激活酶是热胁迫下变性的主要蛋白。因此，热胁迫期间激活酶活性的丧失是由于该蛋白对热变性异常敏感所致，并且部分导致了Rubisco的失活。
