Micro-colorimetric determination of cholinesterase activity of motor end plates in the rat diaphragm

Abstract

A spectrophotometric method is described for the determination of total cholinesterase activity in groups of 3–30 end plates, after staining with copper thiocholine and dissecting from the rat diaphragm, by continuous recording of the yellow colour (412 mμ) produced by reaction between dithiobisnitrobenzoate (1mm) and thiol groups liberated enzymatically from acetylthiocholine (5mm) at pH 7·0. Cholinesterase activity of end plates after correction for muscle ranged from 3·3–22·3 times 10−11m/end plate/hr and muscle cholinesterase activity ranged from 8·8–29·4 times 10−11 μg/hr. 2·9–26·7% of measured end plate cholinesterase activity was attributable to muscle. The results for end plate cholinesterase agreed with those obtained by other workers using microgasometric techniques. It was calculated that there were approximately 3·6 times 106 cholinesterase active sites/end plate which compared closely with 6 times 106 molecules acetylcholine released/ nerve ending/impulse and 2·6 times 106 cholinergic receptors/end plate given in the literature. It is suggested that each acetylcholine molecule after liberation from a nerve ending may interact with one receptor and be destroyed by one cholinesterase active site.