Purification of the phosphorylated night form and dephosphorylated day form of phosphoenolpyruvate carboxylase from Bryophyllum fedtschenkoi.
Purification of the phosphorylated night form and dephosphorylated day form of phosphoenolpyruvate carboxylase from Bryophyllum fedtschenkoi.
复制标题
从苔藓植物中纯化磷酸烯醇丙酮酸羧化酶的磷酸化夜间形式和去磷酸化日间形式。
作者：
G. Nimmo;H. G. Nimmo;I. D. Hamilton;C. A. Fewson;M. Wilkins
Bryophyllum fedtschenkoi的磷酸烯醇式丙酮酸羧化酶被证明以两种形式存在：一种是夜间形式，其被磷酸化并且对苹果酸的抑制具有低敏感性，而一种是白天形式，其被去磷酸化并且对苹果酸的敏感性高10倍。白天和夜间形式的酶纯化保留其不同的苹果酸敏感性和磷酸化状态。纯化的酶含有一个主要蛋白质（亚基Mr 112，000）和一个次要蛋白质（亚基Mr 123，000）。这两种多肽似乎具有密切相关的氨基酸序列，并且在快速制备的提取物中以相似的比例存在。存在于酶的夜形式中的磷酸与丝氨酸共价结合。它不是催化中间体。碱性磷酸酶在体外去除了磷酸基团，并增加了酶对苹果酸的敏感性。白天和夜间的形式的酶可能是四聚体，其表观先生降低了苹果酸的存在下，但不受Mg 2+离子，EDTA，pH值的上升或酶浓度的10倍变化。在白天和晚上制备的叶提取物中观察到的苹果酸敏感性的快速丧失被证明是由于酶的蛋白水解。它在苹果酸存在下和通过酶的磷酸化而减慢。
Phosphoenolpyruvate carboxylase of Bryophyllum fedtschenkoi was shown to exist in two forms: a night form, which is phosphorylated and has low sensitivity to inhibition by malate, and a day form, which is dephosphorylated and 10 times more sensitive to malate. The day and night forms of the enzyme were purified retaining their distinct malate sensitivities and phosphorylation states. The purified enzymes contained a major protein (subunit Mr 112,000) and a minor protein (subunit Mr 123,000). The two polypeptides appeared to have closely related amino acid sequences and were present in a similar ratio in extracts that had been prepared rapidly. The phosphate present in the night form of the enzyme was covalently bound to serine. It was not a catalytic intermediate. Alkaline phosphatase removed the phosphate group in vitro and increased the malate sensitivity of the enzyme to that observed for the day form. Both the day and night forms of the enzyme were probably tetramers, and their apparent Mr was lowered by the presence of malate, but was unaffected by Mg2+ ions, EDTA, a rise in pH or a 10-fold change in enzyme concentration. The rapid loss of malate sensitivity, observed in extracts of leaves prepared during the day and at night, was shown to be due to proteolysis of the enzyme. It was slowed in the presence of malate and by phosphorylation of the enzyme.