Novel allosteric sites on human erythrocyte acetylcholinesterase identified by two monoclonal antibodies.
Monoclonal antibodies against human erythrocyte acetylcholinesterase (acetylcholine acetylhydrolase EC 3.1.1.7) have been examined for inhibition of enzyme activity. Of sixteen antibodies analyzed, only one (C1B7) inhibited enzyme activity, indicating selection of an unusual susceptible site. The inhibitory activity of C1B7 was characterized and compared to another inhibitory antibody, AE-2, previously described by Fambrough et al. (Proc. Natl. Acad. Sci. USA 79, 1078, 1982). Maximal demonstrated inhibition was 84% for C1B7 and 72% for AE-2 and antibody inhibition of enzyme activity was equivalent for the reduced and alkylated acetylcholinesterase monomer and the intact dimer. The Ki (stoichiometry of the enzyme-antibody reaction estimated from enzyme kinetics) was 1.0 for C1B7 and 4.8 molecules of antibody per monomer of acetylcholinesterase for AE-2. The antibodies did not compete with one another for binding to acetylcholinesterase, indicating that they have different target epitopes on the enzyme. Antibody binding to the enzyme was not specifically affected by any of the anticholinesterase agents tested: (a) the irreversible esteratic site-directed inhibitor diisopropylfluorophosphate; (b) the reversible active site-directed inhibitors edrophonium, neostigmine, BW284c51, and carbachol; and (c) allosteric site-directed compounds propidium and gallamine. Kinetic analysis of their effects provide evidence that both antibodies decrease the catalytic rate of enzyme activity and have little or no effect on substrate binding.
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
C E
CE
Olson
V
V
Chhajlani
J T
JT
August
E D
ED
Schmell
D000110:Acetylcholinesterase / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification; D000495:Allosteric Site; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002846:Chromatography, Affinity; D000939:Epitopes / Q000032:analysis; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007700:Kinetics
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D000110:Acetylcholinesterase