A protease that nicks the approximately 150-kilodalton (kDa) single-chain type A botulinum neurotoxin into the approximately 150-kDa di-chain form in vitro was isolated from Clostridium botulinum type A (Hall strain) cultures. The di-chain neurotoxin generated in vitro is composed of an approximately 50-kDa light chain and an approximately 100-kDa heavy chain which are disulfide linked and is indistinguishable from the di-chain neurotoxin that forms in vivo and is routinely isolated (M.L. Dekleva and B.R. DasGupta, Biochem. Biophys. Res. Commun. 162:767-772, 1989). This enzyme was purified greater than 1,000-fold by ammonium sulfate precipitation, QAE-Sephadex Q-50, Sephadex G-100, and CM-Sephadex C-50 chromatography steps with the synthetic substrate N-benzoyl-DL-arginine-p-nitroanilide. The approximately 62-kDa amidase (protease) is a complex of 15.5- and 48-kDa polypeptides (determined by polyacrylamide gel electrophoresis) that could not be separated without sodium dodecyl sulfate. The enzyme has an isoelectric point of pH 5.73, a pH optimum of 6.2 to 6.4, an absolute requirement for a thiol-reducing agent as well as a divalent metallic cation (probably Ca2+) for activity, and a temperature optimum of 70 degrees C. Tests with several synthetic substrates indicated the high specificity of the enzyme for arginyl amide bonds.

Clostridium botulinum synthesizes the type A botulinum neurotoxin (NT) as a ∼ 150 kDa single chain protein. Post-translational proteolytic processing yields a ∼ 150 kDa dichain protein composed of a ∼ 50 kDa light and ∼ 100 kDa heavy chain, which has higher toxicity. Trypsin's action mimics the endogenous proteolytic processing [12]. The proteolytic cleavages could occur at 4 sites. We have examined 2 such sites and defined the peptide sequences before and after proteolytic processing. The N-terminal residues of the newly synthesized ∼ 150 kDa single chain NT, ProPheValAsnLys-, remain intact at the N-terminus of the ∼ 50 kDa light chain generated either in the clostridial culture or in vitro with trypsin or with a protease purified from the homologous bacterial culture [10]. The clostridial protease cleaves the single chain NT in vitro, at 13 the distance from its N-terminus, on the amino side of Gly of the sequence -GlyTyrAsnLysAlaLeuAsnAspLeu- before cleaving the bond LysAla at a slower rate. The data indicate that the dichain NT is formed in the bacterial culture in at least 2 steps. Cleavage at XGly produces a ∼ 100 kDa heavy chain-like fragment which is then truncated; cleavage 4 residues downstream at LysAla, and excision of the tetrapeptide GlyTyrAsnLys, generates the mature heavy chain with Ala as its N-terminal residue. The ∼ 100 kDa heavy chain generated in vitro, by nicking the single chain NT with trypsin, also has AlaLeuAsn- as the N-terminal residues.

Botulinum neurotoxin (NT) serotype A isolated from cells from young cultures (∼8 h) of Clostridiumbotulinum type A is a ∼150 kDa single chain protein. Supernatant from older cultures (96 h) yields ∼150 kDa dichain NT composed ∼50 and ∼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 13rd 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 >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, invitro 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.

Botulinum neurotoxin (NT) has two distinct structural regions called L and H chains (~50 and ~100 kDa, respectively). Although the H chain is responsible for binding of the NT to neuronal cells, it is not known which of the subunits is internalized and therefore responsible for causing the blockage of acetylcholine release in susceptible neuronal cells. In this report we describe for the first time the preparation of type A NT which is selectively radiolabeled at either the L or the H chain subunit. Such NT preparations will be useful as tools for determining the distribution of L and H chains in poisoned neuronal cells and the role that each subunit plays in inducing toxicity. The L and H chains of the NT (~150 kDa) were separated, purified, and then individually radiolabeled by reductive methylation of the lysine residues using [3H]- or [14C]formaldehyde. The labeled L and H chains were reconjugated with the complementary unlabeled L and H chains. Formation of SS and noncovalent bonds between the L and H chains regenerated the ~150 kDa NT. Autoradiographs of sodium dodecyl sulfate polyacrylamide gels confirmed that each reconstituted NT preparation was labeled at only one subunit chain. NT selectively labeled at either the L or the H chain had specific radioactivities of ca. 25–30 and 45–55 μCi/μmol, respectively, and toxicity (mouse LD50/mg protein) values of 2.2 ± 1.1 × 107 and 3.0 ± 1.0 × 107, respectively. A linear increase in the specific radioactivity of L and H chain subunits was observed with increasing concentrations of 3H- or 14C-labeled formaldehyde in the reaction mixture and with increasing concentrations of L or H chain in the reaction mixture.