Munday M R, Hardie D G
Eur J Biochem. 1984 Jun 15;141(3):617-27. doi: 10.1111/j.1432-1033.1984.tb08237.x.
Three cyclic AMP-independent acetyl-CoA carboxylase kinases (A, B1 and B2) have been isolated from lactating rat mammary gland, using phosphocellulose chromatography, high performance gel filtration, and affinity chromatography on casein-Sepharose and phosvitin-Sepharose. These protein kinases have been identified with previously described kinases by the following criteria. Kinase A phosphorylates the same sites on rabbit mammary acetyl-CoA carboxylase as acetyl-CoA carboxylase kinase 2, which was originally described as a contaminant of rabbit mammary acetyl-CoA carboxylase purified by the poly(ethylene glycol)procedure. Kinase A will henceforth be referred to as acetyl-CoA carboxylase kinase-2. Kinase B1 has been identified with casein kinase II by its heparin sensitivity, elution behaviour on phosphocellulose, molecular mass, substrate specificity and subunit composition. Kinase B2 has been identified with casein kinase I by its elution behaviour on phosphocellulose, molecular mass, substrate specificity and subunit composition. The three kinases phosphorylate distinct sites on acetyl-CoA carboxylase. Phosphorylation by either casein kinase I or II does not affect enzyme activity. However, acetyl-CoA carboxylase kinase 2 inactivates acetyl-CoA carboxylase reversibly, in an identical manner to cyclic-AMP-dependent protein kinase, and phosphorylates sites located on identical peptides. Acetyl-CoA carboxylase kinase-2 can, however, be distinguished from the free catalytic subunit of cyclic-AMP-dependent protein kinase by its molecular mass, its substrate specificity, its elution behaviour on phosphocellulose, and its complete lack of sensitivity to the protein inhibitor of cyclic-AMP-dependent protein kinase. We also present evidence that phosphorylation of acetyl-CoA carboxylase by cyclic-AMP-dependent protein kinase occurs directly and not via a bicyclic cascade system as proposed by other laboratories.
利用磷酸纤维素色谱法、高效凝胶过滤法以及酪蛋白 - 琼脂糖亲和色谱法和卵黄高磷蛋白 - 琼脂糖亲和色谱法，已从泌乳大鼠乳腺中分离出三种不依赖环磷酸腺苷（cAMP）的乙酰辅酶A羧化酶激酶（激酶A、激酶B1和激酶B2）。通过以下标准，已将这些蛋白激酶与先前描述的激酶进行了鉴定。激酶A使兔乳腺乙酰辅酶A羧化酶上的相同位点磷酸化，与乙酰辅酶A羧化酶激酶2相同，后者最初被描述为通过聚乙二醇方法纯化的兔乳腺乙酰辅酶A羧化酶的污染物。激酶A此后将被称为乙酰辅酶A羧化酶激酶 - 2。激酶B1已通过其对肝素的敏感性、在磷酸纤维素上的洗脱行为、分子量、底物特异性和亚基组成与酪蛋白激酶II进行了鉴定。激酶B2已通过其在磷酸纤维素上的洗脱行为、分子量、底物特异性和亚基组成与酪蛋白激酶I进行了鉴定。这三种激酶使乙酰辅酶A羧化酶上的不同位点磷酸化。酪蛋白激酶I或II的磷酸化均不影响酶活性。然而，乙酰辅酶A羧化酶激酶2以与依赖环磷酸腺苷的蛋白激酶相同的方式可逆地使乙酰辅酶A羧化酶失活，并使位于相同肽段上的位点磷酸化。然而，乙酰辅酶A羧化酶激酶 - 2可通过其分子量、底物特异性、在磷酸纤维素上的洗脱行为以及对依赖环磷酸腺苷的蛋白激酶的蛋白抑制剂完全不敏感，与依赖环磷酸腺苷的蛋白激酶的游离催化亚基区分开来。我们还提供了证据表明，依赖环磷酸腺苷的蛋白激酶对乙酰辅酶A羧化酶的磷酸化是直接发生的，而不是如其他实验室所提出的通过双环级联系统发生。
