In Vivo Phosphorylation: Development of Specific Antibodies to Detect the Phosphorylated PEPC Isoform for the C4 Photosynthesis in Zea mays
In Vivo Phosphorylation: Development of Specific Antibodies to Detect the Phosphorylated PEPC Isoform for the C4 Photosynthesis in Zea mays
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体内磷酸化：开发特异性抗体来检测玉米 C4 光合作用的磷酸化 PEPC 同工型
DOI：
10.1007/978-1-4939-9865-4_18
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Izui Katsura
中科院分区：
文献类型：
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作者：
Ueno Yoshihisa;Yoshizawa-Kumagaye Kumiko;Emura Junji;Urabe Tomoko;Yoshiya Taku;Furumoto Tsuyoshi;Izui Katsura
磷酸烯醇式丙酮酸羧化酶（PEPCs）是一类在C4光合作用中起着初始CO2固定作用的酶，在维管植物中受可逆磷酸化的调节。PEPC分子上的磷酸化位点不仅在同种型中而且在植物物种中是保守的。抗磷酸肽抗体是检测磷酸化靶蛋白的常用和强有力的工具，具有高特异性。我们产生了两种抗体，一种针对含有磷酸丝氨酸的肽（磷酸肽），另一种针对含有磷酸丝氨酸模拟物（S）-2-氨基-4-膦酰基丁酸（膦酰基肽）的肽。该肽的氨基酸序列取自玉米PEPC的C4-同种型的N-末端区域附近的磷酸化位点周围的位点。前一种抗体几乎特异性地检测PEPC的磷酸化C4-同种型，而后一种抗体对各种植物物种中的磷酸化PEPC具有更广泛的特异性。本文描述了以下程序：（1）磷酸肽和膦酰基肽的制备;（2）兔抗体的制备和纯化;（3）从叶制备细胞提取物，用于用抗体分析PEPC磷酸化;和（4）所获得的抗体的表征。最后，（5）两个案件涉及这些抗体的应用。
Phosphoenolpyruvate carboxylases (PEPCs), mostly known as the enzymes responsible for the initial CO2fixation during C4photosynthesis, are regulated by reversible phosphorylation in vascular plants. The phosphorylation site on a PEPC molecule is conserved not only among isoforms but also across plant species. An anti-phosphopeptide antibody is a common and powerful tool for detecting phosphorylated target proteins with high specificity. We generated two antibodies, one against a peptide containing a phosphoserine (phosphopeptide) and the other against a peptide containing a phosphoserine mimetic, (S)-2-amino-4-phosphonobutyric acid (phosphonopeptide). The amino acid sequence of the peptide was taken from the site around the phosphorylation site near theN-terminal region of the maize C4-isoform of PEPC. The former antibodies detected almost specifically the phosphorylated C4-isoform of PEPC, whereas the latter antibodies had a broader specificity for the phosphorylated PEPC in various plant species. The following procedures are described herein: (1) preparation of the phosphopeptide and phosphonopeptide; (2) preparation and purification of rabbit antibodies; (3) preparation of cell extracts from leaves for analyses of PEPC phosphorylation with antibodies; and (4) characterization of the obtained antibodies. Finally, (5) two cases involving the application of these antibodies are presented.