Isolation of cholinergic receptor protein(s) from Torpedo nobiliana by affinity chromatography

Abstract

The acetylcholine receptor (AChR) was purified 60-fold from the electroplax membranes of Torpedo nobiliana by affinity chromatography utilizing the principal neurotoxin of Naja naja siamensis as ligand. Displacement of the AChR from the Sepharose-bound toxin-AChR complex was far more difficult than when the complex was free in solution, suggesting that covalent linkage of the toxin to the Sepharose significantly changes the ligand-protein interaction. The purified AChR binds one molecule of neurotoxin per ca. 80,000 daltons of protein and shows bands corresponding to molecular weights T-he selective isolation and purification of biologically important macromolecules by "affinity chromatography" exploit the unique biological property of these proteins to bind ligands specifically and reversibly (Cuatrecasas et al., 1968). Elapid snake venoms contain polypeptide neurotoxins which possess the extraordinary properties of binding with very high affinity and with nearly total specificity to the nicotinic acetylcholine receptor (AChR)1 *found in excitable membranes at neuromuscular junctions (Boquet et al., 1966;Lee and Chang, 1966). Hence these neurotoxins appear to be ideal ligands for the isolation and purification of the AChR via affinity chromatography. However, since the interaction between the toxin and AChR was generally regarded as irreversible (Miledi et al., 1971;Meunier et al., 1972), other ligands, notably quaternary nitrogen functions, were used in initial attempts to purify the AChR protein. The use of these selective adsorbants achieved extensive purification of the AChR protein from Electrophorus electricus (Olsen et al., 1972), Narcine entemedor (Schmidt and Raftery, 1972), and Torpedo californica (Schmidt and Raftery, 1973). In each case, these purified preparations were inhomogeneous upon gel electrophoresis which suggested that the preparations were either contaminated with inert proteins or that more than one AChR was present in the tissue.The recent observation that mice given sublethal doses of neurotoxins (0.8-0.9 X LDioo) displayed severe dyspnea and yet recovered within a few hours (Karlsson et al., 1971) was interpreted as being inconsistent with an irreversible interaction between the toxin and AChR. Consequently, an affinity column, using as ligand the principal neurotoxin of Naja naja siamensis, was employed to isolate the AChR (Karlsson el al., 1972) from extracts of Torpedo marmorata. The free AChR was eluted from the column with carbamylcholine and two protein fractions, each of which retained high binding capacity for curare and the neurotoxin, were obtained. As was evident from the affinity chromatography pattern, these fractions were inhof From the

Subunit Structure of α‐Bungarotoxin Binding Component in Mouse Brain

“…Diiodo-toxin of specific activity 1,700 Cilmmol was used. The toxin used for affinity chromatography was purified from the venom of Nuja nuju kuotrthiu (Sigma, St. Louis, Missouri) by chromatography through two columns of SP-Sephadex C-25 by the method of Ong and Brady (1974). The purified toxin gave one band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the results of amino acid analysis were in agreement with the data of Karlsson et al (1971), except for proline.…”

Large‐Scale Purification of the Acetylcholine‐Receptor Protein in Its Membrane‐Bound and Detergent‐Extracted Forms from Torpedo marmorata Electric Organ

“…Several bands have also been found by various authors with their purified fractions of acetylcholine receptor from Torpedo marmorata [35 -371, T. nobiliana [33], T . ocellata [34] and T. californica [19,23,32,38,39].…”

Nicotinic acetylcholine receptor contains multiple binding sites: evidence from binding of alpha-dendrotoxin.

“…a-Ntx was purified from Naja naja siamensis venom (Biotoxins, St. Cloud, FL) as described by Ong and Brady (29). The peptide composition of the column fractions was assessed by NaDodSO4/polyacrylamide gel electrophoresis (30) using slab gels containing exponential or linear gradients of acrylamide from 15 to 22.5%.…”