MASS ISOFORM OF PHOSPHOENOLPYRUVATE CARBOXYLASE FROM THE S. MINUTUM

在单细胞绿色新月藻中， 磷酸烯醇式丙酮酸羧化酶（PEPC）作为两类 具有不同动力学性质的同种型。 PEPC 1为400 kD 同源四聚体，而PEPC 2、PEPC 3和PEPC 4较大（> 1000 kD） 含有102 kD PEPC 1催化亚基的蛋白复合物。 PEPC催化亚基与不相关多肽的关联 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物PEPC之间。 PEPC 2构成 S.微小的 当这个 在金属蛋白酶存在下纯化蛋白复合物 抑制剂，分子量约为130 kD的多肽 （p130），是存在于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 对P130。 免疫印迹分析显示，p130和PEPC 催化亚单元在所有四个色谱过程中精确地共纯化 纯化步骤，表明p130之间的紧密相互作用 和高分子量PEPC同种型中的PEPC。 进一步分析 p130在体外被蛋白激酶A磷酸化， 以及来自S.分钟 工作是 目前正在利用质谱法鉴定p130 肽质量图分析和测序。

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.