Separation, purification, partial characterization and comparison of the heavy and light chains of botulinum neurotoxin types A, B, and E.
Clostridium botulinum produces botulinum neurotoxin (NT) in antigenically distinct forms. When isolated from bacterial cultures type E is a single chain, type B is a mixture of single and two-chain molecules, and type A is essentially a two-chain molecule (Mr approximately 150,000). Protease(s) in the cultures or trypsin nick single-chain NT to the two-chain form. The heavy (Mr approximately 100,000) and light (Mr approximately 50,000) chains of the two-chain molecule remain held together by -S-S-bond(s). The two chains are presumed to have different functions. NT binds to nerve cells via the heavy chain and then light chain enters the cell and blocks release of acetylcholine (Simpson, L. L. (1981) Pharmacol. Rev. 33, 155-188). We nicked single-chain NT to form the two-chain form with trypsin, minimizing secondary cleavages, then separated and purified the heavy and light chains using ion-exchange chromatography. The technique, with minor modifications, is a generalized method for types A, B, and E. These subunit chains (each a single band in sodium dodecyl sulfatepolyacrylamide gel electrophoresis) were analyzed for their complete amino acid compositions. The amino acid contents of the heavy and light chains agreed well with the parent two-chain molecule. This affirms that NT is composed of two chains. The two subunit chains are now usable for amino acid sequence and other studies. Comparison of the amino acid contents indicates more similarity among the light chains than the heavy chains of the three NT types, a similarity that agrees with our published partial amino acid sequences (first 13-18 residues) of these chains. Several (up to 9) different amino acid residues of the heavy chain (which is twice the size of the light chain) are present in double the number of corresponding residues in the light chain.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001905:Botulinum Toxins / Q000302:isolation & purification*; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008970:Molecular Weight; D009498:Neurotoxins / Q000302:isolation & purification*
D000596:Amino Acids; D004220:Disulfides; D046911:Macromolecular Substances; D009498:Neurotoxins; D001905:Botulinum Toxins