Ekdahl K N, Ekman P
J Biochem. 1984 Apr;95(4):917-24. doi: 10.1093/oxfordjournals.jbchem.a134719.
After partial chromatographic purification of rat liver cell sap on DEAE-cellulose, including the removal of type M2 pyruvate kinase, different forms of type L pyruvate kinase were separated by chromatofocusing. Three fractions of pyruvate kinase activity were found, eluting at pH 5.0, 5.2, and 5.3, respectively. The first one was identified as phosphorylated and the second one as unphosphorylated pyruvate kinase. There were strong indications that the third fraction represented a proteolytically modified form of the enzyme, since it co-migrated with a form modified in vitro and had a similarly increased apparent Km for phosphoenolpyruvate. To rule out the possibility of this being a phosphorylated form of pyruvate kinase, the enzyme was incubated with a phosphoprotein phosphatase and then phosphorylated with cAMP-dependent protein kinase. The enzyme was not phosphorylated, like pyruvate kinase modified with subtilisin or calcium-activated protease. There is some evidence that a proteolytically modified pyruvate kinase exists in vivo. This enzyme form has not previously been demonstrated in cell sap, prior to exposure to proteolytic enzymes. The relative amounts of the three forms were determined in livers from starved rats and rats fed on a normal or a carbohydrate-rich diet.
在对大鼠肝细胞液进行部分色谱纯化（包括去除M2型丙酮酸激酶）后，通过色谱聚焦法分离出不同形式的L型丙酮酸激酶。发现了三个丙酮酸激酶活性组分，分别在pH 5.0、5.2和5.3处洗脱。第一个组分被鉴定为磷酸化的丙酮酸激酶，第二个为未磷酸化的丙酮酸激酶。有强烈迹象表明，第三个组分代表该酶的一种经蛋白水解修饰的形式，因为它与体外修饰的一种形式共迁移，并且对磷酸烯醇丙酮酸的表观Km同样增加。为了排除这是丙酮酸激酶磷酸化形式的可能性，将该酶与一种磷蛋白磷酸酶一起孵育，然后用依赖于cAMP的蛋白激酶进行磷酸化。该酶没有被磷酸化，就像用枯草杆菌蛋白酶或钙激活蛋白酶修饰的丙酮酸激酶一样。有一些证据表明，一种经蛋白水解修饰的丙酮酸激酶存在于体内。在暴露于蛋白水解酶之前，这种酶形式以前在细胞液中未被证实。测定了饥饿大鼠以及喂食正常或富含碳水化合物饮食的大鼠肝脏中这三种形式的相对含量。
