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PURIFICATION AND CHARACTERIZATION OF PROTEIN KINASE C FROM A HIGHER PLANT (Brassica campestris L.) AND ANALYSIS FOR ITS BIOCHEMICAL ROLE.

高等植物 (Brassica Campestris L.) 中蛋白激酶 C 的纯化和表征及其生化作用分析。

基本信息

批准号：
05454131
负责人：
NANMORI Takashi
金额：
$ 3.2万
依托单位：
KOBE 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

Protein kinase C (PKC) was partially purified from Brassica campestris L., by successive chromatographies on DEAE-cellulose membrane, hydroxyapatite and pheny1-5PW columns. The purified preparation showed typical characteristics of the conventional type of mammalian PKC that responds to Ca^<2+>, phosphatidylserine, and diacylglycerol or the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. The plant PKC activity was apparently associated with a 75-kDa polypeptide that was recongnized by an antibody against the catalytic domain of rat PKC.Substrate specificity of the plant PKC was similar to that of the rat PKC.A synthetic peptide corresponding to residues 4-14 of myelin basic protein, which is a selective substrate for the mammalian PKC,was phosphorylated efficiently by the plant PKC.These results indicate the existence of a PKC equivalent in higher plant cells.The plant PKC activity was mainly detected in the leaf and the root of this plant. A PKC was also partially puri … More fied from the plant leaf according to the procedures described above.It was well-known that protein phosphortlayion and dephosphorylation are important events during signal transductions in mammalian cells. On the other hand, in plant cells, metabolic regulation by phosphorylation/dephosphorylation have not been elucidated before recent years. Nitrate reductase (NR), which is the key enzyme of nitrate assimilation in higher plant, seems to be one of the enzymes regulated by phosphorylation and dephosphorylation. Our co-workers showed that the NR in Brassica campestris leaf is phosphorylated in vivo in response to environmental light conditions.The PKC fractions partially purified from the plant leaf were allowed to react to the NR purified from the leaf with the presence of gamma-^<32>PATP as phosphorus donor. SDS-PAGE and autoradiography analysis revealed that the NR was phosphorylated only when the Ca2+, phosphatidylserine, and diacylglycerol were present in the reaction mixtures. The result support the idea that the plant PKC involved in phosphorylation of the NR protein. Further studies would be need to clearify the PKC involevement in the regulation of NR activity in response to environmental conditions. In summary of this Grant-in Aid for Scientific Research, we point out not only that PKC homologues exist in the higher plant but also that they have the important role during the plant signal transduction process. Less

蛋白激酶C（PKC）是从白菜中部分纯化的，在DEAE-纤维素膜、羟基磷灰石和Phenyl-5 PW柱上进行连续色谱分离。纯化后的蛋白质显示出哺乳动物PKC的典型特征，它对Ca^2+、磷脂酰丝氨酸、甘油二酯或促肿瘤的佛波酯佛波醇12-肉豆蔻酸酯13-乙酸酯有反应。植物蛋白激酶C的活性与一个75 kDa的多肽有关，该多肽可被大鼠蛋白激酶C催化区的抗体识别，其底物特异性与大鼠蛋白激酶C相似，是哺乳动物蛋白激酶C的选择性底物，结果表明，在高等植物细胞中存在着一种PKC等价物，植物PKC活性主要存在于叶和根中。PKC也被部分纯化， ...更多信息众所周知，蛋白质磷酸化和去磷酸化是哺乳动物细胞信号转导过程中的重要事件。另一方面，在植物细胞中，通过磷酸化/去磷酸化的代谢调节在最近几年之前还没有被阐明。硝酸还原酶（NR）是高等植物硝酸盐同化的关键酶，是受磷酸化和去磷酸化调控的酶之一。我们的合作者发现油菜叶片中的NR在体内响应于环境光条件而磷酸化，允许从植物叶片中部分纯化的PKC组分与从叶片中纯化的NR在存在γ-^<32>-PATP作为磷供体的情况下反应。SDS-PAGE和放射自显影分析表明，NR磷酸化只有当Ca 2+，磷脂酰丝氨酸，和甘油二酯存在于反应混合物中。这一结果支持了植物PKC参与NR蛋白磷酸化的观点。PKC在NR活性调节中的作用有待于进一步研究。在总结本次科研资助项目的基础上，指出PKC同源物不仅存在于高等植物中，而且在植物信号转导过程中起着重要作用。少