Solubilization and characterization of membrane-bound choline O-acetyltransferase in brain

The enzyme choline 0-acetyltransferase (EC 2.3.1.6, ChAT) catalyzes the synthesis of the neurotransmitter acetylcholine in central and peripheral cholinergic nerve tissue. Traditionally, ChAT was thought to exist exclusively as a soluble, cytoplasmic enzyme in nerve endings. However, several experimental results suggest that a form of ChAT exists which is more closely associated with the membranes of nerve endings. For example, although acetylcholine is stored in both the cytoplasm and the vesicles of nerve endings, these two pools are metabolized independently and supply acetylcholine for different modes of release. Cytoplasmic acetylcholine has never been demonstrated to be transported into synaptic vesicles, but choline formed from hydrolyzed acetylcholine may be transported into vesicles for synthesis of vesicular acetylcholine.

The objective of this study was to determine whether membrane-bound forms of ChAT exist, of which one form may be more closely associated with the vesicles. Three fraction of ChAT were solubilized from a crude vesicular membrane fraction of brain, by washing the membranes with three solutions of increasing chaotrophic nature: 100 mM sodium phosphate buffer, pH 7.4 (NaP), 500 mM NaCl, and 2% Tritón DN-65. A variety of biochemical, anatomical, biophysical, developmental, and pharmacological properties of the three fractions were then examined to determine whether they differ from each other. NaP soluble ChAT, which is traditionally thought to be a soluble (cytoplasmic) enzyme, differed from the other two fractions of ChAT with respect to pH profile, heat inactivation, cardiolipin inhibition, hydrophobic chromatography on phenyl-Sepharose, and developmental pattern. NaCl soluble ChAT differed from the other two fractions with respect to K for choline, pH profile, anatomical distribution, sensitivity to septal-hippocampal lesion, heat inactivation, hydrophobic chromatography on phenyl-Sepharose, developmental pattern, and the effect of atropine administration. Tritón soluble ChAT differed from the other two fractions with respect to ability to acetylate homocholine, heat inactivation, and developmental pattern.

The NaP soluble forra of ChAT may supply the cytoplasmic pool of acetylcholine, while it appears that the Tritón soluble form of ChAT may synthesize vesicular acetylcholine. The physiological role of NaCl soluble ChAT is more uncertain, but the results support the possibility that this form may be a precursor for one or both of the other two forms of ChAT.