Neurochemical Research (2) 323-325 (1977)

C o m m e n t

"IMMUNOHISTOCHEMICAL LOCALIZATION OF CHOLINE ACETYLTRANSFERASE:

REAL OR ARTEFACT?" A Reply

L.-P. CHAO, 1 F. J. WOLFGRAM, 1 AND L. F. ENG 2 aDepartment of Neurology

Reed Neurological Research Center UCLA School of Medicine

Los Angeles, California 90024 2Laboratory Service

Veterans Administration Hospital Palo Alto, California 94304

A c c e p t e d D e c e m b e r 28, 1976

One of our main purposes in studying the purified choline acetyltransfer- ase (ChAc) is to localize this enzyme in the nervous system and map the cholinergic tracts. In order to achieve this goal, ChAc from bovine caudate nuclei has been purified (1) and its homogeneity demonstrated beyond doubt (see below). The presence of ChAc in the ventral horn motor neurons of bovine spinal cord has been identified by the immuno- fluorescent technique (2). Recently, positive staining was confirmed not only in the cell bodies of ventral horn motor neurons of either bovine or rabbit spinal cords but also in the primary cortex (area 4) and in the medullary and granular layers of rabbit cerebellum (3) using the peroxi- dase-antiperoxidase immunochemical method (4). The authenticity of these immunochemical findings is supported by an independent study of

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3 2 4 CHAO ET AL.

the distribution of ChAc in rabbit cerebellum with a microenzymatic analysis (5). All these data support the conclusion that the antigen (ChAc) is pure and its microscopic localization is now possible.

In an unusual paper in which no new data were presented, Rossier (6) cast doubt on the validity of our work on the purification of choline acetyltransferase (1) and on our localization of this enzyme in nervous tissue by immunofluorescence (2). We comment on Rossier's paper specifically as follows:

Rossier states (paragraph 4) "pure antigen from vertebrates is not yet available." The homogeneity of our ChAc preparation from bovine caudate nuclei has been established by polyacrylamide gel electrophore- sis at different pH's and gel concentrations (1) and also by immunologi- cal criteria (2). The homogeneity of the enzyme was further demon- strated by the presence of a single amino terminal and a single carboxyl terminal residue and also by the fact that specific cleavage of the molecule at the methionine or tryptophan residues gave the number of peptides predicted from the amino acid analysis of the intact molecule (7). Rossier's observation (paragraph 5) about the inhomogeneity of his purified ChAc preparation in SDS gels is irrelevant, since ChAc aggre- gates as well as dissociates in SDS (2,7), thus making it impossible to use SDS electrophoresis as a criterion of homogeneity.

The data that Rossier presented (in his Table 1) on the specific activities of the various ChAc preparations are erroneous and mislead- ing. First of all, the specific activities of enzyme preparations obtained from different tissue sources and assayed by different methods cannot be compared as criteria of homogeneity. In Rossier's assay of his enzyme preparation, only (14C)-acetyl CoA was used (6) while in our assay system (1,8) 0.215 nmol of (14C)-acetyl CoA was diluted with 61.73 nmol of unlabeled acetyl CoA. After correcting the dilution factor of the unlabeled acetyl-CoA, the specific activity of our final purified prepara- tion is about 420 nmol/min/mg (1).

In a similar manner, studies on the inhibition of ChAc by its antisera cannot be compared unless carried out under identical conditions. We found (2) a 30-40% inhibition of ChAc when it was incubated for 20 h with its antiserum. Matsuda et al. (9) reported that the maximum inhibition (50%) of glutamic acid decarboxylase by its antiserum oc- curred at 5 days' incubation, and this enzyme has been successfully localized by immunohistochemistry (10).

In summary, we feel that the charges of Rossier--that our preparation of ChAc is not homogenous, that it is of low specific activity, and that it is not inhibited enough by its antiserum to be localized by immunoflu- orescence--are groundless. It is unfortunate that Rossier has not at-

LOCALIZATION OF CHOLINE ACETYLTRANSFERASE 325

tempted to reproduce our results but rather attacks them to explain his failure to purify the enzyme by another method.

REFERENCES

1. CHAO, L.-P., and WOLFGRAM, F. 1973. Purification and some properties of choline acetyltransferase from bovine brain. J. Neurochem. 20:1075-1081.

2. ENG, L. F., UYEOA, C, T., CHAO, L.-P., and WOLFGRA~, F. 1974. Antibody to bovine choline acetyltransferase and immunofluorescent localization of the enzyme in neurones. Nature 250:243-245.

3. KAN, K.-S., CHAO, L.-P., WOLFGt~AM, F., and EN6, L. F. 1977. Immunohistochemi- cal localization of choline acetyltransferase in CNS. Trans. Am. Soc. Neurochem. 8:171.

4. STERNBERGER, L. A., HARDY, P. H., JR., CUCULIS, J. J., and MEYER, H. G. 1970. The unlabeled antibody enzyme method of immunohistochemistry. J. Histochem. Cytochem. 18:315-333.

5. MCCAMAN, R. E., and HUNT, J. M. 1965. Microdetermination of choline acetylase in nervous tissue. J. Neuroehern. 12:253-259.

6. ROSSlER, J. 1975. Immunohistochemical localization of choline acetyltransferase: real or artefact? Brain Res. 98:619-622.

7. CHAO, L.-P. 1975. Subunits of choline acetyltransferase. J. Neurochem. 25:261-266. 8, CHAO, L.-P., and WOLFGRAM, F. 1972. Spectrophotometric assay for choline acetyl-

transferase. Anal. Biochem. 46:114-118. 9. MATSUDA, T., Wu, J.-Y., and ROBERTS, E. 1973. Immunochemical studies on

glutamic acid decarboxylase from mouse brain. J. Neurochem. 2l: 15%166. 10. SAITO, K., BARBER, R., Wu, J.-Y., MATSUDA, T., ROBERTS, E., and VAUGHN, J. E.

1974. Immunohistochemical localization of glutamate decarboxylase in rat cerebellum. Proe. Natl. Acad. Sci. 71:269-273.