Ethylcholine mustard aziridinium ion (
AF64A, MEChMAz) has been proposed as a cholinergic neuron-specific
neurotoxin. We report that in further studies on its mechanism of action incubation of the
cholinergic neuroblastoma X
glioma cell line, NG-108-15, with 100 microM
AF64A resulted in a rapid decrease in cellular
choline acetyltransferase (ChAT) activity which preceded cytotoxicity. Thus, a 60-85% decrease in ChAT activity was measured within 5 h of
AF64A exposure, whereas cell lysis (measured as the release of the cytosolic
enzyme lactate dehydrogenase into the medium) did not become apparent until 18 h of
AF64A exposure. This led us to examine the effects of
AF64A on partially purified ChAT. We report a concentration- and time-dependent inhibition of partially purified ChAT by
AF64A that could not be reversed by dialysis but could be prevented by coincubation of the
enzyme and
AF64A with
choline but not with
acetyl-coenzyme A. We present kinetic evidence that
choline and
AF64A compete for the same site on the
enzyme. In addition,
thiosulfate, which inactivates the aziridinium ion, eliminated
AF64A's capacity to inhibit the
enzyme.
AF64A also irreversibly inhibited partially purified
choline kinase and
acetylcholinesterase but not
lactate dehydrogenase,
alcohol dehydrogenase,
carboxypeptidase A, or
chymotrypsinogen,
enzymes that do not use
choline as a substrate or product. Thus, the data suggest that
AF64A acts as an irreversible active site directed inhibitor of ChAT and possibly other
enzymes recognizing
choline.