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

通过DEAE-纤维素膜、羟基磷灰石和苯基1-5PW柱层析，从白菜中分离得到部分蛋白激酶C(PKC)。纯化的制剂表现出典型的哺乳动物PKC的典型特征，它对钙、磷脂酰丝氨酸和二酰甘油或促进肿瘤的佛波酯12-肉豆蔻酸13-醋酸酯有反应。植物PKC的活性明显与一种被抗大鼠PKC催化结构域的抗体识别的75 kDa多肽有关。植物PKC的底物特异性与大鼠PKC相似。哺乳动物PKC的选择性底物髓鞘碱性蛋白的第4-14位残基对应的合成肽被植物PKC有效地磷酸化。这些结果表明，高等植物细胞中存在与PKC等价物。植物PKC活性主要在植物的叶和根中检测到。蛋白激酶C也是部分纯化的…。众所周知，蛋白质的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/去磷酸化来调节代谢的机制直到最近几年才被阐明。硝酸还原酶(NR)是高等植物硝酸盐同化的关键酶，似乎是受磷酸化和去磷酸化调节的酶之一。我们的同事们发现，甘蓝型油菜叶片中的NR在体内被磷酸化，对环境光条件做出反应。从植物叶片中部分纯化的PKC组分被允许与从叶片中纯化的NR在γ-^&lt；32&GT；PATP作为磷供体的存在下发生反应。SDS-PAGE和放射自显影分析表明，只有当反应混合物中存在Ca~(2+)、磷脂酰丝氨酸和二酰甘油时，NR才被磷酸化。这一结果支持了植物PKC参与NR蛋白磷酸化的观点。需要进一步的研究来明确PKC参与调节NR活性以响应环境条件。综上所述，我们指出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具有重要作用。较少

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