Department of Psychiatry, Institute of Psychiatric Research, Indiana University School of Medicine, IN, USA.
J Neurochem. 2012 Aug;122(4):672-80. doi: 10.1111/j.1471-4159.2012.07793.x. Epub 2012 Jul 2.
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 Alzheimer's 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 Alzheimer's disease compared to controls.
胆碱乙酰转移酶（ChAT）在神经末梢中将胆碱和乙酰辅酶 A（AcCoA）催化反应生成乙酰胆碱（ACh）。ACh 代谢在许多生理和不同疾病状态中都有影响，如阿尔茨海默病。因此，一种特异性、敏感性和可靠的检测 ChAT 酶活性的方法在许多情况下都非常有用。我们使用现有的基于放射性核素的酶活性测定法，观察到非胆碱能细胞中可检测到的 ChAT 信号，这表明测定法中的一种污染物会产生虚假信号。先前的报告表明，L-乙酰肉碱（LAC）会污染许多 ChAT 活性测定，因为通过有机提取将 LAC 与 ACh 分离存在困难。为了确定这种假设的伪影的来源并解决该问题，我们开发了一种基于纸层析的测定法，用于检测乙酰胆碱和该测定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且经济的方法来分离和验证可能在大多数实验室中无需特殊设备即可进行的各种反应产物或污染物的身份。我们的第二个目标是将这种分离方法应用于死后的人脑组织样本。我们的测定法成功地检测到了几种污染物，尤其是在使用脑组织的测定中，并且可以将预期的 ACh 产物与这些污染物分离。我们进一步证明，该测定法可用于测量相同样品中的肉碱乙酰转移酶（CrAT）活性，并且比较 ChAT 和 CrAT 的测定表明，CrAT 在神经元组织和神经元细胞培养物中的活性相对于 ChAT 较高。因此，我们描述的这种基于简单层析的测定法可以使用通常可用且廉价的材料来测量从污染物中分离出来的特定反应产物。此外，我们发现与对照相比，阿尔茨海默病患者脑提取物中的 ChAT 活性显著降低。
