Inhibition of choline acetyltransferase by monoclonal antibodies.
Four monoclonal antibodies were obtained to rat brain choline acetyltransferase (CAT). The enzyme was purified 95,000-fold from rat brain by precipitation with acetic acid at pH 4.5, fractionation with 40 to 60% (NH4)2SO4, CM-Sephadex chromatography, and affinity column chromatography on agarose-hexane-coenzyme A. The enzyme preparation was applied to the affinity column in the presence of 10 mM acetylcholine to increase the affinity of CAT for coenzyme A; the enzyme then was eluted with 10 mM acetyl coenzyme A. Fusion of P3X63 Ag8 myeloma cells with spleen cells isolated from a BALB/c mouse that had been immunized with affinity-purified CAT with a specific activity of 29.4 mumol of ACh synthesized/min/mg of protein resulted in the isolation of four hybridomas synthesizing antibodies to CAT that inhibit the activity of the enzyme. Anti-CAT 1 or 2 inhibits CAT activity 100%. At the highest antibody concentration tested, anti-CAT 3 inhibited acetylcholine synthesis 80%. Hybridoma antibody-dependent inhibition of CAT activity was reversed by dissociation of immune complexes via dilution, demonstrating that antibody binding does not irreversibly alter the structure of the enzyme. When bound to [rabbit anti-mouse IgG . protein A Staphylococcus aureus] complexes, anti-CAT 1, 3, and 4 each were effective reagents for the precipitation of CAT activity from solution. Thirty-one to 53% of the precipitated enzyme was recovered following the dissociation of immune complexes. Anti-CAT 1, 2, and 3 inhibit CAT from 18-day chick embryo brain, NS20-Y mouse neuroblastoma cells, and rat brain.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000936:Antigen-Antibody Complex / Q000276:immunology; D001921:Brain / Q000201:enzymology; D002645:Chickens; D002795:Choline O-Acetyltransferase / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000302:isolation & purification; D002845:Chromatography; D005260:Female; D005590:Fractional Precipitation; D006825:Hybridomas / Q000276:immunology; D007163:Immunosorbent Techniques; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats; D011919:Rats, Inbred Strains
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002795:Choline O-Acetyltransferase