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

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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.