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Choline-O-acetyltransferase (ChAT) is the enzyme which catalyses the biosynthesis of the neurotransmitter acetylcholine in cholinergic neurons. Here we show that in mouse cholinergic NS-20Y neuroblastoma cells cultured in the presence of either okadaic acid (serine/threonine phosphatases 1 and 2A inhibitor) or KN-62 (CaM kinase inhibitor) ChAT activity and mRNA either increased or decreased as a function of the drug concentration, respectively. After 24 h exposure, okadaic acid exerted a dramatic effect on cell morphology; cells became round and had no more neurites. On the contrary, KN-62 induced a slight morphological differentiation of the cells. The present results suggest that phosphatases 1 and 2A and CaM kinase could mediate regulation of ChAT gene expression.

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.