一种使用ADP特异性己糖激酶测量激酶和ATP酶动力学的灵敏偶联酶测定法。
A Sensitive Coupled Enzyme Assay for Measuring Kinase and ATPase Kinetics Using ADP-Specific Hexokinase.
作者信息
McFarlane Ciaran R, Murray James W
机构信息
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
Deparment of Life Sciences, Imperial College London, London, United Kingdom.
出版信息
Bio Protoc. 2020 May 5;10(9):e3599. doi: 10.21769/BioProtoc.3599.
Kinases and ATPases perform essential biological functions in metabolism and regulation. Activity of these enzymes is commonly measured by coupling ATP consumption to the synthesis of a detectable product. For most assay systems the ATP concentration during the reaction is unknown, compromising the precision of the assay. Using the ADP-specific hexokinase (ADP-HK) from the thermophilic archaeon the protocol outlined here allows real time coupling of ATP consumption to downstream signal change enabling accurate kinetic measurements. ADP-HK phosphorylates glucose that is then used by glucose-6-phosphate dehydrogenase to reduce NAD to NADH which can be measured at 340 nm. We have shown this assay to be sensitive to the detection of micromole quantities of ADP with no detectable background from ATP.
激酶和ATP酶在新陈代谢和调节过程中发挥着重要的生物学功能。这些酶的活性通常通过将ATP消耗与可检测产物的合成相偶联来测定。对于大多数检测系统而言，反应过程中的ATP浓度是未知的，这会影响检测的精度。利用嗜热古菌中的ADP特异性己糖激酶（ADP-HK），本文所述方案可实现ATP消耗与下游信号变化的实时偶联，从而进行准确的动力学测量。ADP-HK将葡萄糖磷酸化，然后葡萄糖-6-磷酸脱氢酶利用该葡萄糖将NAD还原为NADH，NADH可在340nm处进行检测。我们已经证明该检测方法对微摩尔量ADP的检测敏感，且无ATP产生的可检测背景。
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