MASS ISOFORM OF PHOSPHOENOLPYRUVATE CARBOXYLASE FROM THE S. MINUTUM

来自 S. MINUTUM 的磷酸酚丙酮酸羧化酶的质量亚型

• 批准号: 6258856
• 负责人: JEAN RIVOAL
• 金额: --
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6258856
• 关键词: bioengineering /biomedical engineering; biological products; biomedical resource; biotechnology; technology /technique development

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.

在单细胞绿藻 Selenastrum minutum 中， 磷酸烯醇丙酮酸羧化酶 (PEPC) 以两类形式存在 具有不同动力学特性的亚型。 PEPC1 是 400 kD 同四聚体，而 PEPC2、PEPC3 和 PEPC4 较大 (> 1000 kD) 含有 102 kD PEPC1 催化亚基的蛋白质复合物。 PEPC 催化亚基与无关多肽的关联 似乎是造成动力学特性差异的原因 四聚体和蛋白质复合物 PEPC 之间。 PEPC2 构成 S. minutum 中可提取 PEPC 活性的主要形式。 当这个 在金属蛋白酶存在下纯化蛋白质复合物 抑制剂，一种分子量约为 130 kD 的多肽 (p130)，是复合物中存在的主要多肽之一 PEPC 催化亚基。 产生多克隆抗血清 反对p130。 免疫印迹分析表明 p130 和 PEPC 催化亚基在所有四个色谱过程中精确共纯化 纯化步骤，表明 p130 之间存在紧密相互作用 以及高分子量 PEPC 异构体​​中的 PEPC。 进一步分析 揭示 p130 在体外被蛋白激酶 A 磷酸化，如下所示 以及来自 S. minutum 的内源性蛋白激酶。 工作是 目前正在使用质谱法鉴定 p130 肽质量图分析和测序。