The molecular composition of progenitor toxins produced by a Clostridium botulinum type A strain (A-NIH) was analyzed. The strain produced three types of progenitor toxins (19 S, 16 S, and 12 S) as reported previously. Purified 19 S and 16 S toxins demonstrated the same banding profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating that they consist of the same protein components. The nontoxic components of the 19 S and 16 S toxins are a nontoxic non-hemagglutinin (HA) (molecular mass, 120 kDa) and HA. HA could be fractionated into five subcomponents with molecular masses of 52, 35, 20, 19, and 15 kDa in the presence of 2-mercaptoethanol. The molar ratios of neurotoxins, nontoxic non-HAs, and each HA subcomponent of the 19 S and 16 S toxins showed that only HA-35 of the 19 S toxin was approximately twice the size of that of the 16 S toxin, suggesting that the 19 S toxin is a dimer of the 16 S toxin cross-linked by the 35-kDa subcomponent. The nontoxic non-HA of the 12 S toxin, but not those of the 19 S and 16 S toxins, demonstrated two bands with molecular masses of 106 and 13 kDa on SDS-PAGE with or without 2-mercaptoethanol. It was concluded from the N-terminal amino acid sequences that 106- and 13-kDa proteins were generated by a cleavage of whole nontoxic non-HA. This may explain why the 12 S and 16 S (and 19 S) toxins exist in the same culture. We also found that the HA and its 35-kDa subcomponent exist in a free state in the culture fluid along with three types of progenitor toxins.
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