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 immunoglobulin G) antibody. Adsorption of nerve terminals on to the immunoadsorbent was assessed by using a variety of enzyme markers and gave a maximum yield of 24% of choline acetyltransferase, whereas non-specific binding was less than 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 shown to be intact, osmotically sensitive and metabolically active.

Choline Uptake and Metabolism in Affinity‐Purified Cholinergic Nerve Terminals from Rat Brain

“…In conclusion, it has been shown that the affinity-purified nerve terminal preparation takes up choline as well as synthesizing and releasing acetylcholine under appropriate conditions. Taken together with the previous demonstration of their ability to oxidize glucose (Richardson et al, 1984), these results suggest that such terminals are metabolically competent and constitute a suitable system for the study of cholinergic metabolism.…”

“…~ P. J. RICHARDSON (1984). In brief, after homogenization and centrifugation at 1,000 g for 10 min, the S , fraction (Gray and Whittaker, 1962) was incubated for 40 min with a 1:80 dilution of sheep anti-Chol-I serum, prepared a s previously described (Richardson et al, 1984). The antiserum recognizes a ganglioside antigen (Chol-1 ) localized specifically on the plasma membrane of cholinergic terminals in mammalian brain (Jones et al, 1981;Richardson, 1983).…”

“…glutamate decarboxylase by a method similar to that described by Fonnum et al (1970), and lactate dehydrogenase by the method of Keiding et al (1974). Nerve terminal protein bound to the immunoadsorbent was assayed by the method of Udenfriend et al (1972), as described by Richardson et al (1984). Table I illustrates the susceptibility of labelled choline uptake to inhibitors such as hemicholinium-3 and to low extracellular N a + levels.…”

Adenosine receptor‐mediated modulation of acetylcholine release from rat striatal synaptosomes

“…Immunoaffinity purification of cholinergic nerve terminals Cholinergic nerve terminals were affinity purified from rat striatum using sheep anti-(Chol-1) serum and a mouse anti-(sheep IgG) immunoadsorbent as previously described (Richardson et al, 1984;Richardson, 1986). The yield of cholinergic nerve terminals is expressed as units (1 nmol h-' at 37C) of choline acetyltransferase (EC 2.3.1.6) activity and amounted to approximately 10% of the initial activity present.…”

Purification of caveolae by affinity two-phase partitioning using biotinylated antibodies and NeutrAvidin–dextran

“…Such an approach is facilitated by the increasing availability of antibodies directed against speciWc membrane proteins. Antibodies bound to immunoaYnity beads have been used for the isolation of cells [7,8] and membrane fractions [9][10][11], but their use in aYnity partitioning has not been explored so far.…”