TISSUE DISTRIBUTION OF THE AMP-ACTIVATED PROTEIN-KINASE, AND LACK OF ACTIVATION BY CYCLIC-AMP-DEPENDENT PROTEIN-KINASE, STUDIED USING A SPECIFIC AND SENSITIVE PEPTIDE ASSAY
TISSUE DISTRIBUTION OF THE AMP-ACTIVATED PROTEIN-KINASE, AND LACK OF ACTIVATION BY CYCLIC-AMP-DEPENDENT PROTEIN-KINASE, STUDIED USING A SPECIFIC AND SENSITIVE PEPTIDE ASSAY
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DOI：
10.1111/j.1432-1033.1989.tb15185.x
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发表时间：
1989-12-01
期刊：
影响因子：
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通讯作者：
HARDIE, DG
中科院分区：
文献类型：
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作者：
DAVIES, SP;CARLING, D;HARDIE, DG
我们已经合成了两种肽，一种基于大鼠乙酰辅酶A羧化酶（SSMS肽）上AMP活化蛋白激酶的独特位点（Ser 79）周围的确切序列，另一种是其中对应于环AMP依赖性蛋白激酶（Ser 77）位点的丝氨酸残基被丙氨酸（SAMS肽）取代。这两种肽磷酸化与类似的动力学AMP激活的蛋白激酶，但只有SSMS肽是一个底物环AMP依赖性蛋白激酶。SAMS肽不被测试的其他五种纯化蛋白激酶中的任何一种磷酸化。AMP活化蛋白激酶对SAMS肽的Km高于乙酰辅酶A羧化酶，但肽磷酸化的Vmax是其亲本蛋白的2.5倍。因此，这种肽提供了一种方便和灵敏的AMP活化蛋白激酶的测定。乙酰辅酶A-羧化酶激酶和肽激酶活性copurify通过6个步骤，从大鼠肝脏的postmitochondrial上清液，表明SAMS肽是一个特定的底物AMP激活的蛋白激酶在这个组织中。我们不能证明AMP依赖性的激酶活性在粗制剂，显然是由于内源性AMP保持绑定的酶。然而，8-溴腺苷5-单磷酸（Br 8AMP）是变构（AMP）位点的部分激动剂，并且2 mM Br 8AMP的抑制可用于测试测量AMP刺激形式的激酶。使用这种方法，我们已经检查了9个不同的大鼠组织中的激酶活性，加上小鼠巨噬细胞系，并发现有一个组织之间的相关性表达显着水平的肽激酶活性和那些活跃在合成或储存的脂质。我们还使用肽测定表明，环AMP依赖性蛋白激酶不激活纯化的AMP激活的蛋白激酶，并不影响部分纯化的AMP激活的蛋白激酶的内源性激酶激酶的激活。
We have synthesized two peptides, one based on the exact sequence around the unique site (Ser79) for the AMP-activated protein kinase on rat acetyl-CoA carboxylase (SSMS peptide) and another in which the serine residue corresponding to the site for cyclic-AMP-dependent protein kinase (Ser77) was replaced by alanine (SAMS peptide). Both peptides were phosphorylated with similar kinetics by the AMP-activated protein kinase, but only the SSMS peptide was a substrate for cyclic-AMP-dependent protein kinase. The SAMS peptide was not phosphorylated by any of five other purified protein kinases tested. The Km of AMP-activated protein kinase for the SAMS peptide is higher than that for acetyl-CoA carboxylase, but the Vmax for peptide phosphorylation is 2.5 times higher than that of its parent protein. This peptide therefore gives a convenient and sensitive assay for the AMP-activated protein kinase. Acetyl-CoA-carboxylase kinase and peptide kinase activities copurify through six steps from a postmitochondrial supernatant of rat liver, showing that the SAMS peptide is a specific substrate for the AMP-activated protein kinase in this tissue. We could not demonstrate AMP-dependence of the kinase activity in crude preparations, apparently due to endogenous AMP remaining bound to the enzyme. However, 8-bromoadenosine 5-monophosphate (Br8AMP) is a partial agonist at the allosteric (AMP) site, and inhibition by 2 mM Br8AMP can be used to test that one is measuring the AMP-stimulated form of the kinase. Using this approach, we have examined the kinase activity in nine different rat tissues, plus a mouse macrophage cell line, and find that there is a correlation between tissues expressing significant levels of peptide kinase activity and those active in the synthesis or storage of lipids. We also use the peptide assay to show that cyclic AMP-dependent protein kinase does not activate purified AMP-activated protein kinase, and does not affect the activation of partially purified AMP-activated protein kinase by endogenous kinase kinase.