Chromatographic separation of reaction products from the choline acetyltransferase and carnitine acetyltransferase assay: differential ChAT and CrAT activity in brain extracts from Alzheimer's disease versus controls
Chromatographic separation of reaction products from the choline acetyltransferase and carnitine acetyltransferase assay: differential ChAT and CrAT activity in brain extracts from Alzheimer's disease versus controls
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DOI：
10.1111/j.1471-4159.2012.07793.x
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发表时间：
2012-08-01
影响因子：
4.7
通讯作者：
Lahiri, Debomoy K.
中科院分区：
文献类型：
--
作者：
Bailey, Jason A.;Lahiri, Debomoy K.
神经化学杂志。（2012）122，672680中所述。胆碱乙酰转移酶（ChAT）在神经末梢催化胆碱与乙酰辅酶A（AcCoA）反应生成乙酰胆碱（ACh）。乙酰胆碱代谢在许多方面的生理和不同的疾病状态，如阿尔茨海默病的影响。因此，一个具体的，敏感的，可靠的方法检测ChAT酶活性是在许多情况下的巨大效用。使用现有的基于放射性核素的酶活性测定，我们已经观察到可检测的ChAT信号从非胆碱能细胞，这表明在测定中的污染物产生一个人为的信号。以往的报道表明，L-乙酰肉碱（LAC）污染的ChAT活性的许多测定，因为很难从乙酰胆碱分离LAC的有机萃取。为了确定这种假设的伪影的来源并纠正问题，我们开发了一种基于纸层析的检测方法，用于检测乙酰胆碱和该检测方法的其他污染反应产物，包括LAC。我们的第一个目标是开发一种简单而经济的方法，用于解析和验证各种反应产物或污染物的身份，该方法可以在大多数实验室中进行，而无需专门的设备。我们的第二个目标是将这种分离方法应用于死后人脑组织样本。我们的检测成功地检测到几种污染物，特别是在使用脑组织的检测，并允许从这些污染物中分离出预期的乙酰胆碱产品。我们进一步证明，该测定可用于测量肉毒碱乙酰转移酶（CrAT）在相同的样品中的活性，并比较ChAT和CrAT的测定表明，CrAT是高度活跃的神经元组织和神经元细胞培养物相对于ChAT。因此，我们描述的简单的基于色谱的测定允许使用常用且廉价的材料测量与污染物分离的特定反应产物。此外，我们表明，ChAT活性显着降低，从阿尔茨海默病的脑提取物相比，控制。
J. Neurochem. (2012) 122, 672680. Abstract Choline acetyltransferase (ChAT) catalyzes the reaction between choline and acetylcoenzyme A (AcCoA) to form acetylcholine (ACh) in nerve terminals. ACh metabolism has implications in numerous aspects of physiology and varied disease states, such as Alzheimers disease. Therefore a specific, sensitive, and reliable method for detecting ChAT enzyme activity is of great utility in a number of situations. Using an existing radionuclide-based enzyme activity assay, we have observed detectable ChAT signals from non-cholinergic cells, suggesting a contaminant in the assay producing an artifactual signal. Previous reports have suggested that L-acetylcarnitine (LAC) contaminates many assays of ChAT activity, because of difficulties in separating LAC from ACh by organic extraction. To determine the source of this hypothesized artifact and to rectify the problem, we have developed a paper chromatography-based assay for the detection of acetylcholine and other contaminating reaction products of this assay, including LAC. Our first goal was to develop a simple and economical method for resolving and verifying the identities of various reaction products or contaminants that could be performed in most laboratories without specialized equipment. Our second goal was to apply this separation method in postmortem human brain tissue samples. Our assay successfully detected several contaminants, especially in assays using brain tissue, and allowed the separation of the intended ACh product from these contaminants. We further demonstrate that this assay can be used to measure carnitine acetyltransferase (CrAT) activity in the same samples, and assays comparing ChAT and CrAT show that CrAT is highly active in neuronal tissues and in neuronal cell cultures relative to ChAT. Thus, the simple chromatography-based assay we describe allows the measurement of specific reaction products separated from contaminants using commonly available and inexpensive materials. Further, we show that ChAT activity is significantly reduced in brain extracts from Alzheimers disease compared to controls.