RT Journal Article
SR Electronic
T1 Inhibition of choline acetyltransferase by monoclonal antibodies
JF The Journal of Neuroscience
JO J. Neurosci.
FD Society for Neuroscience
SP 175
OP 180
DO 10.1523/JNEUROSCI.05-01-00175.1985
VO 5
IS 1
A1 WL Strauss
A1 M Nirenberg
YR 1985
UL http://www.jneurosci.org/content/5/1/175.abstract
AB 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.