Membrane binding characteristics of choline-o-acetyltransferase in rat hippocampal nerve terminals

Choline-0-acetyltransferase (ChAT; EC 2.3.1.6.) is the enzyme which catalyzes the formation of the excitory neurotransmitter acetylcholine. Although most of the ChAT in central cholinergic nerve terminals is soluble, some is also non-ionically associated with membranes. The major objective of this dissertation research was to determine how this membrane-bound detergent soluble form of ChAT (D-ChAT) was anchored to membranes in rat hippocampal nerve terminals. I tested the hypothesis that D-ChAT was anchored to membranes by a glycosylphosphatidylinositol (GPI) anchor. ChAT appeared to possess three characteristics common to many GPI-anchored proteins: (1) phosphatidylinositol specific-phospholipase C (PI-PLC) selectively released it from membranes, (2) PI-PLC treatment converted it from a detergent soluble into an aqueous form, and (3) an antibody to an epitope on the GPI anchor (anti-CRD) immunoreacted with the cytosolic form of ChAT (S3-ChAT) on Western blots. I also tested the possibility that D-ChAT might be one of the first intracellular GPI-anchored proteins described by internalizing PI-PLC into synaptosomes and determining if it would release D-ChAT from membranes into the cytosol. Internalized PI-PLC increased the amount of ChAT found in the cytosol, while reducing the amount of D-ChAT associated with membranes. In some tissues, endogenous glycosyl-phosphatidylinositol-specific phospholipase Cs (GPI-PLCs) function to release GPI-anchored proteins from membranes. To determine if an endogenous GPI-PLC might function to release D-ChAT from membranes, I utilized the GPI-PLC inhibitor zinc. Zinc inhibited an endogenous temperature-dependent release of ChAT from rat hippocampal minces, an endogenous release of ChAT from intracellular membranes into the cytosol of synaptosomes, and an endogenous conversion of ChAT from a detergent into an aqueous form in a plasma membrane enriched subcellular fraction (P4). These results suggest that some of the D-ChAT in rat hippocampal nerve terminals is GPI-anchored intracellularly, and that an endogenous GPI-PLC-like enzyme releases it from membranes into the cytosol.