Concanavalin A-Sepharose Affinity Chromatography of Axon Plasma Membrane Proteins. Heterogeneity of Cholinergic Binding Sites |
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Authors: | Judith K Marquis Dana C Hilt Henry G Mautner |
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Institution: | Department of Biochemistry and Pharmacology, Tufts University School of Medicine, Boston, Massachusetts 02111.U.S.A. |
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Abstract: | Abstract: Lysolecithin-solubilized proteins from axon plasma membranes of lobster walking leg nerve bundles were chromatographed on concanavalin A (Con A)-sepharose. Bound glycoproteins were eluted with α-methyl-D- mannoside. Near quantitative recovery of total protein was observed, 20–30% of the total protein being eluted in the Con A-binding glycoprotein fraction. A 5-fold enrichment of acetylcholinesterase (AChE) activity was achieved, demonstrating the glycoprotein nature of the axonal enzyme. The chromatographed fractions were characterized for binding of 3H]quinuclidinyl benzilate (QNB), 3nicotine (Nic), and 1251]α-bung arotoxin (BgTx) in an attempt to distinguish possible "muscarinic" and "nicotinic" binding sites in axonal membranes. All of the high-affinity "muscarinic" 3H]QNB binding activity appeared in the non-Con A-binding protein fractions, while binding of the two "nicotinic" ligands, 3Nic and 125I-BgTx, was found in both the glycoprotein and non-Con A-binding protein fractions. BgTx interaction with the Con A-binding glycoproteins could be blocked with dtubocurarine, but BgTx binding in the non-Con A-binding proteins was not inhibited by curare. The significance of multiple cholinergic binding sites in axonal membranes is discussed. These data suggest a closer similarity between the cholinergic ligand binding proteins of peripheral nerve membrane and ganglia than between the axonal cholinergic binding sites and the ACh receptor of the neuromuscular junction. |
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