PHOSPHORYLATION OF RAT-BRAIN CHOLINE-ACETYLTRANSFERASE AND ITS RELATIONSHIP TO ENZYME-ACTIVITY

Choline acetyltransferase catalyzes the formation of acetylcholine fro m choline and acetyl-CoA in cholinergic neurons. The present study exa mined conditions for modulation of kinase-mediated phosphorylation of this enzyme. By using a monospecific polyclonal rabbit anti-human chol ine acetyltransferase antibody to immunoprecipitate cytosolic and memb rane-associated subcellular pools of enzyme from rat hippocampal synap tosomes, we determined that only the cytosolic fraction of the enzyme (67,000 +/- 730 daltons) was phosphorylated under basal, unstimulated conditions. The quantity of this endogenous phosphoprotein was depende nt, in part, upon the level of intracellular calcium, with P-32(i) inc orporation into the enzyme in nerve terminals incubated in nominally c alcium-free medium only 43 +/- 7% of control. The corresponding enzyma tic activity of cytosolic choline acetyltransferase did not appear to be altered by lowered cytosolic calcium, whereas membrane-associated c holine acetyltransferase activity was decreased to 58 +/- 11% of contr ol. Depolarization of synaptosomes with 50 muM veratridine neither alt ered the extent of phosphorylation or specific activity of cytosolic c holine acetyltransferase, nor induced detectable phosphorylation of me mbrane-associated choline acetyltransferase, although the specific act ivity of the membrane-associated enzyme was increased to 132 +/- 5% of control. In summary, phosphorylation of choline acetyltransferase doe s not appear to regulate cholinergic neurotransmission by a direct act ion on catalytic activity of the enzyme.