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

在本研究中，我们试图从日本电鳐的电器官中纯化ChAT，制备一种针对鱼的ChAT的抗体，用于免疫组织化学染色鱼胆碱能神经。根据Fonnum（1969）的描述对ChAT活性进行监测，并经Ryan和McClure（1979）修改，纯化程序基本上根据Ryan和McClure（1979）完成。把两条鱼的二百克电器官用硫酸铵沉淀溶解和分馏。在原方法中用哺乳动物大脑进行醋酸沉淀的过程中省略了，因为在这一步骤中ChAT的活性大部分丢失。在羧基甲基-Sepharose C-50离子交换层析、辅酶A (CoA)-Sepharose亲和柱和羟基磷灰石上进行了进一步的纯化。在CM-Sepharose柱中，原方法使用pH为7.2的缓冲液，酶不被柱吸收。通过使用不同pH的缓冲液进行测试，可以清楚地看到，在pH为6.8时，凝胶吸收了大部分ChAT。以0.2M NaCl洗脱酶。在亲和柱中以0.6 ~ 0.8 m NaCl洗脱ChAT。在羟基磷灰石柱中，酶不仅具有硫酸铵梯度，而且具有NaCl梯度。两种组分均含有一种分子量为68000的主蛋白，但均不均匀。在家兔身上获得抗血清，并用于金鱼脊髓的免疫组织化学。然而，抗血清对运动神经元没有特异性。由此得出结论，鱼类胆碱能神经的纯化与哺乳动物胆碱能神经的纯化一样困难，需要开发其他染色胆碱能神经的方法。这一定是针对ChAT的单克隆抗体或针对ACh本身的多克隆抗体的产生。

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.