Purification and characterization of a bungarotoxin polypeptide which blocks nicotinic receptor function in primary culture of adrenal chromaffin cells.
In primary cultures of bovine chromaffin cells, commercially available preparations of alpha-bungarotoxin inhibit the acetylcholine (ACh)- or nicotine-evoked release of endogenous catecholamines. The potency of different lots of alpha-bungarotoxin is not related to the alpha-bungarotoxin peptide content but to that of another peptide (termed P-4 bungarotoxin) present as an impurity in the alpha-bungarotoxin preparations. P-4 Bungarotoxin was isolated and purified to homogeneity by high-pressure liquid chromatography (HPLC). Homogeneity was established by a variety of means, including polyacrylamide gel electrophoresis, HPLC, end carboxy group analysis and NH2-terminal amino acid sequence. Purified P-4 bungarotoxin contains approximately 121 amino acid residues, and it is different in its amino composition, molecular weight, and amino acid sequence from alpha-bungarotoxin and beta-bungarotoxin. P-4 Bungarotoxin (IC50 congruent to 1 nM) blocked the ACh-induced release of endogenous catecholamines but failed to block the KCl-induced catecholamine release. Although P-4 bungarotoxin is endowed with phospholipase A2 activity, its effect on ACh-evoked catecholamine release persists when the phospholipase activity is blocked (99.9%) by treatment of the toxin with p-bromophenacyl bromide. P-4 Bungarotoxin may represent a useful tool with which to study nicotinic receptor function in sympathetic and central nervous system neurons.
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D002038:Bungarotoxins / Q000494:pharmacology*; D002395:Catecholamines / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D002838:Chromaffin System / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D009538:Nicotine / Q000494:pharmacology; D011978:Receptors, Nicotinic / Q000187:drug effects*; D013329:Structure-Activity Relationship
D002038:Bungarotoxins; D002395:Catecholamines; D011978:Receptors, Nicotinic; D009538:Nicotine; D000109:Acetylcholine