Metabolism of acetylcholine in the nervous system of Aplysia californica. III. Studies of an indentified cholinergic neuron

[3H] choline and [3H] acetyl CoA were injected into the cell body of an identified cholinergic neuron, the giant R2 of the Aplysia abdominal ganglion, and the fate and distribution of the radioactivity studied. Direct eveidence was obtained that the availabliity of choline to the enzymatic machinery limits synthesis. [3H] choline injected intrasomatically was converted to acetylcholine far more efficiently than choline taken up into the cell body from the bath. Synthesis from injected [3H] acety CoA was increased more than an order of magnitude when the cosubstrate was injected together with a saturating amount of unlabeled choline. In order to study the kinetics of acetylcholine synthesis in the living neuron, we injected [3H] choline in amounts resulting in a range of intracellular concentrations of about four orders of magnitude. The maximal velocity was 300 pmol of acetylcholine/cell/h and the Michaelis constant was 5.9 mM [3H] choline; these values agreed well with those previously reported for choline acetyltransferase assayed in extracts of Aplysia nervous tissue. [3H] acetylcholine turned over within the injected neuron with a half-life of about 9 h. The ultimate product formed was betaine. Subcellular distribution of [3H] acetylcholine was studied using differential and gradient centrifuagtion, gel filtration, and passage through cellulose acetate filters. A small portion of acetylcholine was contained in particulates the size and density expected of cholinergic vesicles.