Botulinum neurotoxin type E: studies on mechanism of action and on structure-activity relationships.
Single chain type E botulinum neurotoxin was isolated from culture fluids of Clostridium botulinum (strain Alaska E-43). The neurotoxin, which migrated as a single band in polyacrylamide gel electrophoresis with sodium dodecylsulfate, had a molecular weight of approximately 147,000. Single chain type E neurotoxin that was exposed to trypsin was converted to a dichain molecule. Pretreatment of the single chain molecule with 1,2-cyclohexanedione, a reagent that selectively modifies arginine residues, inhibited trypsin-induced generation of the dichain molecule. In dose-response experiments (10(-13) to 10(-9) M) on the isolated neuromuscular junction (phrenic nerve-hemidiaphragm preparation), the dichain neurotoxin was approximately two orders of magnitude more potent than the single chain neurotoxin. Neither specie of neurotoxin (1 pmol/mouse, in vivo; 1 X 10(-11) M, in vitro) was very effective in blocking autonomic transmission (vagus nerve-atrium preparation). The neuromuscular blocking action of the dichain molecule was divided into a sequence of three steps. There was an initial binding step that was relatively rapid, little influenced by temperature and which left the neurotoxin partially accessible to the neutralizing effects of antitoxin. There was a translocation step that was temperature dependent, antagonized by ammonium chloride and methylamine hydrochloride and which caused the neurotoxin to become inaccessible to the neutralizing effects of antitoxin. Finally, there was an intracellular lytic step, during which the toxin blocked excitation-secretion coupling.
D000818:Animals; D001341:Autonomic Nervous System / Q000187:drug effects; D001711:Biotransformation; D001904:Botulinum Antitoxin / Q000494:pharmacology; D001905:Botulinum Toxins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D006224:Cricetinae; D003512:Cyclohexanones / Q000494:pharmacology; D004058:Diffusion; D006168:Guinea Pigs; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D009469:Neuromuscular Junction / Q000187:drug effects; D009498:Neurotoxins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D013045:Species Specificity; D013329:Structure-Activity Relationship; D009435:Synaptic Transmission / Q000187:drug effects; D014357:Trypsin / Q000378:metabolism
D001904:Botulinum Antitoxin; D003512:Cyclohexanones; D009498:Neurotoxins; C018031:1,2-cyclohexanedione; D014357:Trypsin; D001905:Botulinum Toxins