Preparation of antibodies specific to choline acetyltransferase from bovine caudate nucleus and immunohistochemical localization of the enzyme.
Choline acetyltransferase (CATase; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6) has been purified from bovine caudate nucleus. The specific activity of the pure enzyme was 120-160 mumol of acetylcholine formed per mg per min. The purified enzyme separated into two bands (band A and band B) when electrophoresed in a pH 4.3 gradient gel. Both bands exhibited CATase activity. Antisera were prepared in rabbits to each form. Immunotitrations using either antisera resulted in quantitative precipitation of CATase activity from enzyme preparations at all stages of purification. Both antisera produced single immunoprecipitin lines in double-diffusion experiments when run against the enzyme at each stage of purity. Immunoprecipitin lines cut from double-diffusion gels contained CATase activity, demonstrating that the observed reaction was due to antibody interaction with the enzyme. Immunoelectrophoresis also showed a single immunoprecipitin line against the purified enzyme as well as against a crude caudate extract. These results indicated that immunochemically pure and specific antisera have been prepared to bovine CATase and that both the A and B forms of the enzyme have common antigenic sites. The antisera were utilized to localize the enzyme in bovine brain. The localization was exclusively neuronal with both antisera, and both forms of CATase were present in the same population of neurons. Subtle differences in the precise intracellular distribution of enzyme were observed with the two antisera, indicating that although the two molecular forms of CATase have antigenic sites in common they are not antigenically identical.
D000818:Animals; D000918:Antibody Specificity; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002421:Caudate Nucleus / Q000201:enzymology*; D002795:Choline O-Acetyltransferase / Q000276:immunology* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D008970:Molecular Weight
D002795:Choline O-Acetyltransferase