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Molecular Mechanism of Photoregulation of a Plant Enzyme by Phosphorylation/Dephosphorylation

植物酶磷酸化/去磷酸化光调节的分子机制

基本信息

批准号：
02808034
负责人：
IZUI Katsura
金额：
$ 0.96万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

Phosphoenolpyruvate carboxylase(PEPC)involved in C4 photosynthesis is known to be activated under light conditions and deactivated under dark conditions. This photoregulation is mediated by phosphorylation of PEPC. The study was undertaken to obtain a clue to the molecular mechanism for this process. Previously we found that PEPC of maize(a C4 plant)can be phosphorylated by a mammalian cyclic AMP-dependent protein kinase and is l e dto the activated state similarly to the activation in vivo. Furthermore, we identified the site of phosphorylation to be S e r - 1 4. A protein kinase which phosphorylates PEPC(PEPC-PK)was partially purified from maize green leaves and the site of phosphorylation was shown to be identical with the site phosphorylated by the mammalian protein kinase. A recombinant maize PEPC produced in E. coli cells, which is assumed to be free of phosphorylation was prepared and its kinetic properties were compared with the dark-form PEPC(40% phosphorylated)prepared from maize leaves. Kinetic propertied such as Km for PEP and Ki for malate were not significantly different from one another. To characterize PEPC-PK, a series of PK inhibitors with different specificities were tested and it was found that the enzyme was strongly inhibited by the inhibitor for Ca^<2+>- calmodulin-dependent PK such as myosin light chain kinase. Furthermore, the enzyme was inhibited by EGTA and the inhibition was relieved by the addition of Ca^<2+>. These results strongly suggest that the light signal is transduced to PK by elevating the intracellular concentration of Ca^<2+>. Molecular characterization of PK and cloning of its cDNA is now under progress.

已知参与C4光合作用的磷酸烯醇式丙酮酸羧化酶（PEPC）在光照条件下被激活，在黑暗条件下失活。这种光调节由PEPC的磷酸化介导。进行这项研究是为了获得这一过程的分子机制的线索。以前我们发现玉米（C_4植物）的PEPC可以被哺乳动物环腺苷酸依赖性蛋白激酶磷酸化，并进入类似于体内活化的活化状态。此外，我们确定的磷酸化位点是S er-14。从玉米绿色叶中部分纯化了一种磷酸化PEPC的蛋白激酶（PEPC-PK），其磷酸化位点与哺乳动物蛋白激酶磷酸化位点相同。在E.制备了假定没有磷酸化的大肠杆菌细胞，并将其动力学性质与从玉米叶制备的暗型PEPC（40%磷酸化）进行了比较。动力学特性，如PEP的Km和苹果酸的Ki没有显着差异。为了表征PEPC-PK，测试了一系列具有不同特异性的PK抑制剂，并且发现该酶被Ca^2+-钙调蛋白依赖性PK的抑制剂如肌球蛋白轻链激酶强烈抑制。EGTA对该酶有抑制作用，Ca^<2+>可解除该抑制作用.这些结果有力地表明，光信号是通过升高细胞内Ca^<2+>浓度而转化为PK的。PK的分子表征及其cDNA的克隆目前正在进行中。