Radiochemical micro assays for the determination of choline acetyltransferase and acetylcholinesterase activities.
Radiochemical micro assays for the determination of choline acetyltransferase and acetylcholinesterase activities.
复制标题
用于测定胆碱乙酰转移酶和乙酰胆碱酯酶活性的放射化学微量测定。
作者：
F. Fonnum
1.胆碱乙酰转移酶的检测方法是基于标记的乙酰辅酶A与未标记的胆碱反应生成标记的乙酰胆碱。2.分别用辅酶A、三磷酸腺苷、镁离子和丙酮干鸽肝提取物与[1-(14)C]乙酸钠或[(3)H]乙酸钠反应生成乙酰辅酶A和乙酰辅酶A。3.用酮基四苯基硼钠萃取分离得到[1-(14)C]乙酰胆碱。4.[(3)H]乙酰胆碱用四苯基硼钠沉淀除去[(3)H]乙酸钠中的酮类污染物，然后用酮基四苯基硼钠萃取。5.在氰化钠、EDTA和人工合成的乙酰辅酶A存在下，胆碱乙酰转移酶的活性与原位合成的乙酰辅酶A的活性相近。6.乙酰胆碱酯酶的测定是基于标记的乙酰胆碱形成标记的乙酸酯。用酮基四苯基硼钠萃取，可将标记的乙酰胆碱从醋酸盐中定量除去。7.用中枢神经和周围神经组织标本和纯化的酶对方法进行检验。8.胆碱乙酰转移酶和乙酰胆碱酯酶的空白值与20 ng的活性相对应。和5 ng。分别取自脑组织。
1. The methods for the assay of choline acetyltransferase were based on the reaction between labelled acetyl-CoA and unlabelled choline to give labelled acetylcholine. 2. Both synthetic acetyl-CoA and acetyl-CoA formed from sodium [1-(14)C]acetate or sodium [(3)H]acetate by incubation with CoA, ATP, Mg(2+) and extract from acetone-dried pigeon liver were used. 3. [1-(14)C]Acetylcholine was isolated by extraction with ketonic sodium tetraphenylboron. 4. [(3)H]Acetylcholine was precipitated with sodium tetraphenylboron to remove a ketone-soluble contaminant in sodium [(3)H]acetate and then extracted with ketonic sodium tetraphenylboron. 5. The values of choline acetyltransferase activity obtained in the presence of sodium cyanide or EDTA and synthetic acetyl-CoA were similar to those obtained with acetyl-CoA synthesized in situ. 6. The assay of acetylcholinesterase was based on the formation of labelled acetate from labelled acetylcholine. The labelled acetylcholine could be quantitatively removed from the acetate by extraction with ketonic sodium tetraphenylboron. 7. The methods were tested with samples from central and peripheral nervous tissues and purified enzymes. 8. The blank values for choline acetyltransferase and acetylcholinesterase corresponded to the activities in 20ng. and 5ng. of brain tissue respectively.