MASS ISOFORM OF PHOSPHOENOLPYRUVATE CARBOXYLASE FROM THE S. MINUTUM

在单细胞绿藻Selenastum minutus中， 磷酸烯醇式丙酮酸羧化酶(PEPC)以两种形式存在 具有不同动力学性质的异构体。PEPC1是一个400kD的 同源四聚体而PEPC2、PEPC3和PEPC4较大(&gt；1000kD) 含有102 kD PEPC1催化亚基的蛋白质复合体。 PEPC催化亚基与无关多肽的相关性研究 似乎是造成动力学性质差异的原因 在四聚体和蛋白质复合体PEPC之间。PEPC2构成 微小链霉菌可提取的PEPC活性的主要形式。当这件事 在金属蛋白酶存在下纯化蛋白质复合体 抑制剂，一种分子质量约为130kD的多肽 (P130)，是存在于与之形成的复合物中的主要多肽之一 PEPC催化亚基。制备了多克隆抗血清。 与第130页的对比。免疫印迹分析显示p130和PEPC 在所有四种层析过程中，催化亚基都准确地结合在一起 纯化步骤，提示p130之间存在紧密的相互作用 PEPC为高分子量的PEPC异构体。进一步分析 发现p130在体外被蛋白激酶A磷酸化为 和来自微小链霉菌的内源蛋白激酶。工作是 目前正在使用质谱学鉴定p130 多肽质谱图分析和测序。

In the unicellular green alga Selenastrum minutum, phosphoenolpyruvate carboxylase (PEPC) exists as two classes of isoforms with distinct kinetic properties. PEPC1 is a 400 kD homotetramer whereas PEPC2, PEPC3 and PEPC4 are large (> 1000 kD) protein complexes containing the 102 kD PEPC1 catalytic subunit. Association of the PEPC catalytic subunit with unrelated polypeptides appears to be responsible for the difference in kinetic properties between the tetrameric and protein complex PEPCs. PEPC2 constitutes the major form of extractable PEPC activity in S. minutum. When this protein complex is purified in the presence of metalloprotease inhibitors, a polypeptide with an approximate molecular mass of 130 kD (p130), is one of the major polypeptides present in the complex with the PEPC catalytic subunit. A polyclonal antiserum was generated against p130. Immunoblot analysis showed that p130 and the PEPC catalytic subunit copurify exactly during all four chromatographic steps of the purification, suggesting a tight interaction between p130 and PEPC in the high molecular mass PEPC isoforms. Further analysis revealed that p130 is phosphorylated in vitro by protein kinase A as well as an endogenous protein kinase from S. minutum. Work is currently in progress to identify p130 using mass spectrometry analysis of peptide mass maps and sequencing.