A basic study on immunohistochemical demonstration on cholinergic nervous system in fish.

本研究试图从日本拟银鱼（Narkejaponica）的电器官中纯化ChAT，制备ChAT抗体，用于化学染色鱼类胆碱能神经。如Fonnum（1969）所述，经Ryan和麦克卢尔（1979）修改，监测ChAT活性，纯化步骤基本上根据Ryan和麦克卢尔（1979）进行。将两条鱼的200克电器官溶解并通过用硫酸铵沉淀而分级。省略了使用哺乳动物脑的原始方法中的乙酸沉淀，因为ChAT活性在该步骤中大部分损失。进一步的纯化是通过连续的色谱上的离子交换色谱与羧甲基-琼脂糖C-50，在亲和柱上的辅酶A（CoA）-琼脂糖和羟基磷灰石。在CM-Sepharose柱中，用原方法中pH7.2的缓冲液，酶不被吸附。通过使用不同pH的缓冲液的测试，很明显，大多数ChAT在pH 6.8时被凝胶吸收。酶在约0.2M NaCl的级分处洗脱。ChAT在0.6-0.8M NaCl中在亲和柱上洗脱。在羟基磷灰石柱中，酶既可以通过硫酸铵梯度洗脱，也可以通过NaCl梯度洗脱。这两种组分都含有一种MW为68，000的主要蛋白质，但不是均匀的。用兔抗血清对金鱼脊髓进行免疫组化。但抗血清对运动神经元无特异性。得出结论，鱼类ChAT的纯化是困难的哺乳动物和其他方法染色胆碱能神经必须开发。这必须是ChAT的单克隆抗体或ACh本身的多克隆抗体的生产。

In this study, we had tried to purify ChAT of the electric organ of the electric ray Narke japonica to make an antibody to the fish ChAT for staining fish cholinergic nerves immunohistochemically. The ChAT activity was monitored as described by Fonnum (1969) modified by Ryan and McClure (1979) and purification procedure was done essentially according to Ryan and McClure (1979). Two hundred grams of the electric organ of two fish was solubilized and fractionated by precipitation with ammonium sulfate. Acetic acid precipitation in the original method using the mammalian brain was omitted, because the ChAT activity was lost mostly in this step. Further purification was obtained by sequential chromatography on an ion-exchange chromatography with carboxymethyl-Sepharose C-50, on an affinity column of coenzyme A (CoA)-Sepharose and on a hydroxylapatite. In CM-Sepharose column, the emzyme was not absorbed by the column in the buffer of pH 7.2 used in the original method. By a test using buffers of different pH, it became clear that most ChAT was absorbed by gel at pH 6.8. The enzyme was eluted at fractions of about 0.2M NaCl. ChAT was eluted at 0.6-0.8M NaCl in affinity column. In hydroxylapatite column, the enzyme was not only by ammonium sulfate gradient but also by NaCl gradient. Both the fractions contained one main protein of MW 68,000 but not homogeneous. Anti-serum to the fractions was gained in a rabbit and was used for immunohistochemistry of the goldfish spinal cord. However, the anti-serum was not spesific to motoneurones. It was concluded that purification of fish ChAT was as difficult as that of mammals and other methods to stain cholinergic nerves had to be developed. That must be either the production of monoclonal antibodies to ChAT or polyclonal antibodies to ACh itself.