Hawley Simon A, Fyffe Fiona A, Russell Fiona M, Gowans Graeme J, Grahame Hardie D
Division of Cell Signalling & Immunology, School of Life Sciences, University of Dundee, Dundee, Scotland, UK.
Methods Mol Biol. 2018;1732:239-253. doi: 10.1007/978-1-4939-7598-3_16.
AMP-activated protein kinase (AMPK) is extremely sensitive to cellular stress, so that nonphysiological activation of the kinase can readily occur during harvesting of cells or tissues. In this chapter we describe methods to harvest cells and tissues, and for kinase assays, that preserve the physiological activation status of AMPK as far as possible. Note that similar care with methods of cell or tissue harvesting is required when AMPK function is monitored by Western blotting, rather than by kinase assays. We also describe methods to determine whether compounds that activate AMPK in intact cells do so indirectly by interfering with cellular ATP synthesis or directly by binding to AMPK and, if the latter, whether this occurs by binding at the AMP-binding sites on the γ subunit or at the ADaM site located between the α and β subunits.
AMP激活的蛋白激酶（AMPK）对细胞应激极其敏感，以至于在细胞或组织收获过程中，该激酶很容易发生非生理性激活。在本章中，我们描述了收获细胞和组织的方法以及激酶测定方法，这些方法尽可能保留了AMPK的生理激活状态。请注意，当通过蛋白质免疫印迹法而非激酶测定法监测AMPK功能时，细胞或组织收获方法也需要同样小心。我们还描述了确定完整细胞中激活AMPK的化合物是通过干扰细胞ATP合成间接激活，还是通过与AMPK结合直接激活的方法，以及如果是后者，这是通过结合γ亚基上的AMP结合位点还是位于α和β亚基之间的ADaM位点发生的。
