Direct inhibition of choline acetyltransferase activity by a monoclonal antibody raised against the plasma membrane of cholinergic nerve terminals.
A monoclonal antibody raised against cholinergic synaptosomal plasma membranes isolated from Torpedo electric organ, inhibited completely amphiphilic and hydrophilic choline acetyltransferase (ChAT) activities extracted and separated by using Triton X-114 phase partition of synaptosomes. We tested whether ChAT inhibition was direct or not. We found that the antibody was inhibiting ChAT in preparations of very low purity as well as ChAT that was immunoprecipitated by using a non-inhibitory anti-ChAT polyclonal antibody. Also, inclusion of acetylcoenzyme A at 20 times its Km during incubation of ChAT and antibody, completely prevented ChAT inhibition. This same concentration of the ChAT substrate could significantly but not completely dissociate the complex enzyme-antibody. These results spoke in favour of a direct inhibition of ChAT; the antibody most probably binds to an epitope that may be located at or near the acetylcoenzyme A binding site. The inhibitory effect on hydrophilic and amphiphilic ChAT was dependent on the antibody concentration, but amphiphilic activity required higher concentrations to be affected to the same extent as hydrophilic activity. This was found not only with Torpedo, but also with rat and human ChAT activities. Thus, the antibody appears to be able to distinguish the two forms of ChAT activity.
Eder-Colli
L
L
Department of Pharmacology, Centre Médical Universitaire, Geneva, Switzerland.
Froment
Y
Y
Monsurro
M R
MR
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D001921:Brain / Q000187:drug effects / Q000201:enzymology*; D002795:Choline O-Acetyltransferase / Q000276:immunology*; D006801:Humans; D066298:In Vitro Techniques; D051381:Rats; D011919:Rats, Inbred Strains; D013574:Synaptosomes / Q000187:drug effects / Q000201:enzymology*; D014101:Torpedo
D000911:Antibodies, Monoclonal; D002795:Choline O-Acetyltransferase