Choline acetyltransferase (ChAT), the enzyme which catalyses the biosynthesis of the neurotransmitter acetylcholine, exists in a soluble and membrane‐bound form in cholinergic nerve terminals of different animal species. This study was performed on the enzyme present in Drosophila central nervous system. We show that the two forms of the enzyme have the same apparent molecular weight (75 kDa) when analysed by immunoblotting using an antibody we raised against the recombinant enzyme. According to different authors, membrane‐bound enzyme might be associated with synaptic vesicles or plasma membrane. Subfractionation of Drosophila head homogenates in linear glycerol gradients showed that ChAT does not associate with synaptic vesicles. Analysis of ChAT activity and immunoreactivity showed that two peaks of ChAT were produced. One peak was present in fractions containing soluble components and the other was associated with rapidly sedimenting membranes containing plasma membranes. ChAT in the first peak was mainly hydrophilic. A large proportion of ChAT associated with rapidly sedimenting membranes was amphiphilic. Further fractionation of these membranes by flotation in sucrose gradients showed that membrane‐associated ChAT sedimented in fractions containing plasma membrane marker. Membrane‐bound ChAT was neither solubilized nor converted to hydrophilic enzyme after membrane treatment with 1 m hydroxylamine, suggesting that the enzyme is not palmitoylated and therefore not anchored to membrane through thioester‐linked long chain fatty acid. Partial solubilization of ChAT present on membranes with urea and carbonate suggests that this form of ChAT is a peripheral membrane protein. Carbonate solubilization of membrane‐bound ChAT converted the enzyme from hydrophobic to hydrophilic protein.

Uncovering the way membrane-bound choline acetyltransferase (ChAT) interacts with membranes and with which membrane in cholinergic neurons may help in understanding its role in acetylcholine metabolism. Subfractionation of rat hippocampal synaptosomes aiming to separate synaptic vesicles from plasma membranes shows that membrane-bound ChAT is bound to plasma membrane. Either detergents or urea and alkali can solubilize membrane-bound enzyme. Detergent-solubilized enzyme has a higher sedimentation rate than urea-alkali solubilized or cytosolic ChAT. Once dissociated, membrane-bound ChAT reassociates specifically with cholinergic plasma membranes, a process that was abolished by previous treatment of membranes with trypsin. Cytosolic ChAT behaves similarly. Thus, in cholinergic synaptosomes, ChAT exists as cytosolic and peripheral activity. Cytosolic ChAT generates peripheral enzyme most probably by interacting with a protein of plasma membrane of cholinergic nerve terminals. This "receptor" protein might regulate the amount of membrane-bound ChAT in cholinergic neurons.

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