A continuous microplate assay for sirtuins and nicotinamide-producing enzymes.
A continuous microplate assay for sirtuins and nicotinamide-producing enzymes.
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DOI：
10.1016/j.ab.2009.07.019
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发表时间：
2009-11-01
影响因子：
2.9
通讯作者：
Denu JM
中科院分区：
文献类型：
--
作者：
Smith BC;Hallows WC;Denu JM
依赖于NAD+的蛋白脱乙酰酶(Sirtuins)和其他产生烟酰胺的酶是许多细胞过程中不可或缺的。然而，目前的活动测量涉及昂贵且耗时的分析。在这里，我们提出了一种光谱分析，它绕过了以前方法的许多问题。这种分析方法允许随着时间的推移对产品进行连续监测，允许确定稳态动力学参数，并且很容易适应高通量筛选。该方法使用了一个酶偶联系统，在这个系统中，烟酰胺被烟酰胺酶转化为烟酸和氨。氨通过谷氨酸脱氢酶转移到α-酮戊二酸，生成谷氨酸，NAD(P)H氧化成NAD(P)+，在340 nm处用分光光度法测定。利用sirtuin-1(Sirt1)和ADP-核糖环化酶CD38的连续测定，得到的稳态动力学参数与其他已发表的方法获得的值非常一致。重要的是，该方法允许用天然乙酰化底物乙酰-辅酶A合成酶-1测定kCAT和Km值，测量哺乳动物细胞提取物中Sirt1、SIRT2和SIRT3的活性，并测定不同Sirt1抑制剂的IC50值。该检测适用于任何烟酰胺形成酶，并将是解决围绕其调节的许多悬而未决的问题的重要工具。
NAD+-dependent protein deacetylases (sirtuins) and other enzymes that produce nicotinamide are integral to many cellular processes. Yet current activity measurements involve expensive and time-consuming assays. Here, we present a spectroscopic assay that circumvents many issues of previous methods. This assay permits continuous product monitoring over time, allows determination of steady-state kinetic parameters, and is readily adaptable to high throughput screening. The methodology uses an enzyme-coupled system in which nicotinamide is converted to nicotinic acid and ammonia by nicotinamidase. The ammonia is transferred to α-ketoglutarate via glutamate dehydrogenase, yielding glutamate and the oxidation of NAD(P)H to NAD(P)+, which is measured spectrophotometrically at 340 nm. Utilizing this continuous assay with sirtuin-1 (Sirt1) and the ADP-ribosyl cyclase CD38, the resulting steady-state kinetic parameters are in excellent agreement with values obtained by other published methods. Importantly, this assay permitted determination of kcat and Km values with the native acetylated substrate acetyl-CoA synthetase-1, measurement of Sirt1, Sirt2, and Sirt3 activities from mammalian cell extracts, and determination of IC50 values of various Sirt1 inhibitors. This assay is applicable to any nicotinamide forming enzyme and will be an important tool to address many outstanding questions surrounding their regulation.
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