Immunoaffinity purification of intact, metabolically active, cholinergic nerve terminals from mammalian brain

Abstract

A method for the immunoaffinity purification of cholinergic nerve terminals from mammalian brain was developed. A sheep antiserum to Torpedo electric organ synaptic membranes, previously shown to be specific for cholinergic terminals in mammalian brain, was incubated with crude mitochondrial fractions prepared from rat brain. Cholinergic nerve terminals sensitized by this serum were purified from the mitochondrial fractions on a high-capacity cellulose immunoadsorbent bearing a mouse monoclonal anti-(sheep IgG) antibody. Adsorption of nerve terminals onto the immunoadsorbent was assessed by using a variety of enzyme markers and gave a maximum yield of 24% of choline acetyltransferase, whereas nonspecific binding was < 1.0% for all of the enzymes measured. Cholinergic terminals were purified 26-fold from rat caudate nucleus, 30-fold from rat hippocampus and 38-fold from rat cerebral cortex. The terminals were intact, osmotically sensitive and metabolically active.