Acetylcholine Esterase Localization

A remarkable discovery in the retinula cells of Mitopus morio was the demonstration, by enzyme histochemistry, of acetylcholine esterase in large numbers of small vesicles. The image below clearly shows dark specks in the vesicles (arrowed) close to the rhabdom (r). These specks are electron-opaque particles of reaction product produced by enzymic splitting of acetylthiocholine in a solution so that copper thiocholine is deposited - the dark specks. Of course, a control is needed to check - see below...

Reaction product localising acetylcholine esterase

Acetylcholine esterase - positive.

Reaction product is present in vesicles (arrowed).

Localizing acetylcholine esterase - reaction product

Verification of enzyme localised as AChE

The incubation medium used for this was the Gomori method, of 1952 with acetylthiocholine as substrate. Here the insoluble electron-dense reaction product is copper thiocholine.
This can be seen in the image on the left.

The activity of AChE can be prevented by the use of the inhibitor 62C47, 1:5-bis-(4-trimethylammoniumphenyl)pentan-3-one di-iodide). This can be used as a control - when no reaction product is produced we can be confident that the enzyme localised in AChE (rather than non-specific esterase).
You can see this in the image on the right.

Acetylcholine esterase - control inhibited by 62C47.

Reaction product is now absent from vesicles (arrowed).

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This information is based on electron microscope observations described in:
Curtis D.J. (1968) Fine structural studies on the eyes of Phalangida. Ph.D. thesis, University of Liverpool.

For further details, see:
Curtis, D.J. (1969) The fine structure of photoreceptors in Mitopus morio (Phalangida). J. Cell Sci., 4: 327-351.

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