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MgCl2
Company: Sigma-Aldrich
Catalog#: 208337-1KG
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A Sensitive Coupled Enzyme Assay for Measuring Kinase and ATPase Kinetics Using ADP-Specific Hexokinase
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2020-05-05
[Abstract]  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 Thermococcus litoralis 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 ... [摘要]  [摘要 ] 激酶和ATPase 在代谢和调节中起着至关重要的生物学功能。通常通过将ATP的消耗与可检测产物的合成耦合来测量这些酶的活性。对于大多数测定系统，反应过程中ATP的浓度是未知的，从而降低了精密度。分析方法。

使用ADP特定己糖激酶（ADP-HK）来自嗜热古菌嗜热litoralis的议定书概述这里可以进行实时耦合ATP消费对下行信号变化进行高精度动态测量。ADP-HK磷酸葡萄糖这随后被葡萄糖-6-磷酸脱氢酶可将NAD + 还原为NADH（可在340 nm处测定），已表明该测定法对检测ADP中的微摩尔量ADP敏感，没有可检测到ATP的背景。

[背景 ] 激酶和ATP酶可通过将ADP的产生与分光光度法可检测的信号偶联来测量。商业供应商提供了试剂盒，用于检测通过将ADP产生的或ATP消耗掉与荧光或生物发光信号的产生进行检测的试剂盒（例如ATP） - 格洛; Promega公司，ADP-传感器; Biovision所属..）这些套件敏感，但很难适用于动力学问题，因为他们是终点分析，测量ADP金额在单个时间点。此外，黑匣子自然这种试剂盒因此很难确定反应混合物是否处于Michaelis-Menten 动力学所需的稳态条件下。

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