alpha-Bungarotoxin immobilized and oriented on a lipid bilayer vesicle surface.
We have developed a new method to assess the binding site on alpha-bungarotoxin (alpha-BGT) for the acetylcholine receptor. It involves the covalent attachment of a palmitic acid chain to the toxin molecule, generating monopalmitoyl-alpha-bungarotoxin (PBGT) which is then immobilized on the surface of a lipid vesicle by a process of spontaneous insertion via the acyl chain into preformed unilamellar vesicles (approximately 800 A in diameter). PBGT itself is able to bind specifically to Triton X-100 solubilized acetylcholine receptors with an association constant, KA, of 5.56 X 10(6) M-1 which is approximately 20-fold lower in affinity than native alpha-BGT. Vesicle-associated PBGT binds to acetylcholine receptor enriched microsac membrane vesicles in aqueous buffer with a KA for both lipid and protein of 4.26 X 10(7) M-1. The putative site of acylation on the PBGT molecule is determined by extensive cleavage of a reduced and carboxymethylated PBGT with thermolysin. An acylated fragment is purified by hydrophobic column chromatography and identified by high-pressure liquid chromatography methods from the known primary sequence of the native toxin as a decapeptide including residues Thr47-Glu56 [C. Y. Lee convention used; see Mebs, D., Narita, K., Iwanaga, S., Samejuma, Y., & Lee, C. Y. (1971) Biochem. Biophys. Res. Commun. 44, 711-716]. Sequential hydrolysis of the fragment from the carboxy terminus with carboxypeptidase Y indicates that Lys51 is the sole site of acylation.(ABSTRACT TRUNCATED AT 250 WORDS)
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004557:Electric Organ / Q000378:metabolism; D007700:Kinetics; D008051:Lipid Bilayers; D008954:Models, Biological; D010446:Peptide Fragments / Q000032:analysis; D011950:Receptors, Cholinergic / Q000378:metabolism*; D014101:Torpedo; D014316:Tritium
D000596:Amino Acids; D002038:Bungarotoxins; D008051:Lipid Bilayers; D010446:Peptide Fragments; D011950:Receptors, Cholinergic; D014316:Tritium