PURIFICATION OF THE AMP-ACTIVATED PROTEIN-KINASE ON ATP-GAMMA-SEPHAROSE AND ANALYSIS OF ITS SUBUNIT STRUCTURE
PURIFICATION OF THE AMP-ACTIVATED PROTEIN-KINASE ON ATP-GAMMA-SEPHAROSE AND ANALYSIS OF ITS SUBUNIT STRUCTURE
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DOI：
10.1111/j.1432-1033.1994.tb19001.x
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发表时间：
1994-07-15
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;HAWLEY, SA;HARDIE, DG
AMP活化的蛋白激酶已通过ATP -γ -琼脂糖凝胶亲和层析法纯化。一部分活性可使用AMP洗脱，而其余部分则使用ATP洗脱。AMP洗脱液包含分子量分别为63、38和35 kDa的三种多肽（p63、p38和p35），其摩尔比（通过考马斯亮蓝结合测定）接近1:1:1。p63先前已被鉴定为AMP结合的催化亚基[卡林，D.；克拉克，P.R.；赞米特，V.A.；哈迪，D.G.（1989年）《欧洲生物化学杂志》186卷，129 - 136页]。这三种多肽在天然凝胶电泳和凝胶过滤中完全共迁移，这表明p38和p35是另外的亚基。通过凝胶过滤估算的斯托克斯半径（5.4 - 5.8 nm）以及通过甘油梯度离心测定的沉降系数（7.9 - 8.4 S）表明，该激酶具有不对称结构，复合物的天然分子量为190 ± 10 kDa。因此，天然酶似乎是一种具有p63/p38/p35（1:1:1）结构的异源三聚体。尽管ATP洗脱液比AMP洗脱液具有更高的比活性（3.5 ± 0.2对2.3 ± 0.2 μmol·min⁻¹·mg⁻¹），但它似乎纯度较低，含有p63、p38和p35以及其他多肽。对蛋白磷酸酶 - 2A和激酶激酶作用的实验研究，以及用抗p63抗体进行的蛋白质印迹分析表明，AMP洗脱液完全处于低活性的去磷酸化形式，而ATP洗脱液是该形式与高活性磷酸化形式的混合物。除了确定AMP活化蛋白激酶的亚基结构外，这些结果还表明，该激酶可通过变构（AMP/ATP）位点或催化（ATP）位点与ATP -γ -琼脂糖凝胶结合，并且激酶激酶的磷酸化作用会增加对后一位点的亲和力。
The AMP-activated protein kinase has been purified by affinity chromatography on ATP-gamma-Sepharose. A proportion of the activity can be eluted using AMP, while the remainder is eluted using ATP. The-AMP eluate contains three polypeptides of 63, 38 and 35 kDa (p63, p38 and p35) in a molar ratio (by Coomassie blue binding) close to 1:1:1. p63 was previously identified as the AMP-binding catalytic subunit [Carling, D., Clarke, P. R., Zammit, V. A. and Hardie, D. G. (1989) Eur J. Biochem. 186, 129-136]. All three polypeptides exactly comigrate both on native gel electrophoresis and on gel filtration, suggesting that p38 and p35 are additional subunits. Estimation of Stokes radius (5.4-5.8 nm) by gel filtration, and sedimentation coefficient (7.9-8.4 S) by glycerol gradient centrifugation, suggest that the kinase has an asymmetric structure with a native molecular mass for the complex of 190 +/- 10 kDa. Thus the native enzyme appears to be a heterotrimer with a p63/p38/p35 (1:1:1) structure. Despite the fact that the ATP eluate has a higher specific activity than the AMP eluate (3.5 +/- 0.2 vs 2.3 +/- 0.2 mu mol min(-1) mg(-1)), it appears to be less pure, containing p63, p38 and p35 plus other polypeptides. Experiments examining the effects of protein phosphatase-2A and kinase kinase, and analysis by Western blotting with anti-p63 antibody, suggests that the AMP eluate is entirely in the low-activity dephosphorylated form, while the ATP eluate is a mixture of that form and the high-activity phosphorylated form. As well as establishing the subunit structure of the AMP-activated protein kinase, these results suggest that the kinase can bind to ATP-gamma-Sepharose through either the allosteric (AMP/ATP) site or the catalytic (ATP) site, and that phosphorylation by the kinase kinase increases the affinity for the latter site.