Synaptosomal "membrane-bound" choline acetyltransferase is most sensitive to inhibition by choline mustard.
The objectives of the present study were to validate the presence of cytoplasmic and membrane-associated pools of choline acetyltransferase (ChAT) in rat brain synaptosomes, and to evaluate inhibition of these different forms of the enzyme by the nitrogen mustard analogue of choline, choline mustard aziridinium ion (ChM Az). The relative distribution of ChAT and lactate dehydrogenase (LDH) was followed in subfractions of synaptosomes to establish whether ChAT activity associated with salt-washed presynaptic membranes represents membrane-bound protein rather than cytosolic enzyme trapped within undisrupted synaptosomes or revesiculated membrane fragments. The percentage of total synaptosomal ChAT activity (14%) recovered in the final membrane pellet always exceeded that of LDH (6%), lending support to the hypothesis that much of the ChAT associated with the membranes was a membrane bound form of the enzyme. Incubation of purified synaptosomes with ChM Az led to irreversible inhibition of ChAT activity; this loss of enzyme activity could not be accounted for by lysis of nerve terminals during incubation in the presence of the mustard analogue. Subfractionation of the ChM Az-treated nerve terminals revealed that the membrane-bound form of ChAT was inhibited to the greatest extent, followed by the ionically membrane-associated enzyme, with the activity of the water-solubilized enzyme not differing significantly from control. Preparation of the synaptosomal ChAT subfractions from untreated nerve terminals prior to incubation with varying concentrations of ChM Az or naphthylvinylpyridine revealed that under these conditions water-solubilized, ionically membrane-associated, and detergent-solubilized membrane-bound pools of ChAT were not differentially inhibited by either compound.(ABSTRACT TRUNCATED AT 250 WORDS)
Rylett
R J
RJ
Department of Physiology, University of Western Ontario, London, Canada.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D002794:Choline / Q000031:analogs & derivatives* / Q000494:pharmacology; D002795:Choline O-Acetyltransferase / Q000032:analysis / Q000037:antagonists & inhibitors*; D003600:Cytosol / Q000201:enzymology; D005260:Female; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D009286:Naphthylvinylpyridine / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013574:Synaptosomes / Q000201:enzymology*
D009286:Naphthylvinylpyridine; C013457:cyclocholine; D007770:L-Lactate Dehydrogenase; D002795:Choline O-Acetyltransferase; D002794:Choline