title: Nicking of single chain Clostridium botulinum type A neurotoxin by an endogenous protease.
abstract: Botulinum neurotoxin (NT) serotype A isolated from cells from young cultures (approximately 8 h) of Clostridium botulinum type A is a approximately 150 kDa single chain protein. Supernatant from older cultures (96 h) yields approximately 150 kDa dichain NT composed approximately 50 and approximately 100 kDa subunits, that remain associated by disulfide and noncovalent bonds. This had led to the assumption that an endogenous protease cleaves a peptide bond at 1/3rd the distance from the N- or C-terminals of the single chain protein. An endogenous protease that causes such a cleavage (nicking) has now been purified greater than 1,000-fold from C. botulinum type A (Hall strain) culture; this culture also produces the single chain NT and eventually yields the dichain NT. The purified protease nicked the pure preparation of single chain type A NT, in vitro at pH 5.6, into a dichain form that was indistinguishable from the dichain NT normally isolated from 96 h cultures. The protease appears specific for nicking serotype A NT because it did not nick single chain serotype B and E NT nor did it enhance toxicity of serotype A, B and E NT.
authors.affiliation: Food Research Institute, University of Wisconsin, Madison 53706.
authors.forename: M L
authors.initials: ML
authors.lastname: Dekleva
authors.forename: B R
authors.initials: BR
authors.lastname: DasGupta
mesh_terms: D000818:Animals; D001905:Botulinum Toxins / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity; D003014:Clostridium botulinum / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D014357:Trypsin / Q000378:metabolism
chemical_list: D010447:Peptide Hydrolases; D014357:Trypsin; D001905:Botulinum Toxins