Botulinum neurotoxin type B. Its purification, radioiodination and interaction with rat-brain synaptosomal membranes

Abstract

1. Neurotoxin from Clostridium botulinumtype B was purified to homogeneity by affinity and ion-exchange chromatography; specific neurotoxicity of this protein (M_r of ∼ 155000) following trypsinisation attained a level of 2 x 10⁸ mouse LD₅₀ units/mg protein. 2. ¹²⁵I-iodination of the toxin to high specific radioactivities (19–63 TBq/mmol) yielded typically >65% of its original toxicity; dodecyl sulphate gel electrophoresis under reducing conditions, after trypsinisation, showed that the larger polypeptide (M_r of ∼ 101 000) was labelled preferentially. 3. Saturable binding of the ¹²⁵I-labelled neurotoxin to rat cerebrocortical synaptosomes was observed and Scatchard analysis showed a low content of acceptors with high affinity (K_d= 0.3 − 0.5 nM; B_max∼ 30–60 fmol/mg protein), together with a much larger population of weak-affinity sites. 4. No significant differences in binding affinity were seen in competition experiments using native or fully activated (trypsinised) neurotoxin, indicating that chain cleavage is not essential for acceptor-toxin interaction. Type A botulinum neurotoxin showed a limited capacity to inhibit the synaptosomal binding of labelled type B toxin, even at high concentrations (1 μM), and other neurotoxins were without effect, emphasising the acceptor selectivity. 5. Near-complete loss of specific toxin binding was produced by preincubation of synaptosomes with neuraminidase whereas inhibition of the low-affinity sites with wheat-germ agglutinin was less pronounced; such inactivation was prevented by inclusion of selective inhibitors (2,3-dehydro-2-deoxy-N-acetylneuraminic acid and N-acetylglucosamine, respectively). These observations implicate N-acetylneuraminic acid and, possibly, other sugar moieties as constituents of the toxin acceptors. 6. Trypsinisation of synaptosomes gave incomplete inhibition of binding when assayed with 1 nM or 10 nM ¹²⁵I-iodinated toxin. Detailed analysis of the actions of neuraminidase, trypsin and heat treatment on the concentration dependence of toxin binding suggest the existence of at least two distinguishable populations of sites that contain N-acetylneuraminic acid, with a protein component being associated with the acceptors of lower affinity. 7. These findings are discussed in relation to those previously reported for type A neurotoxin and to the possible physiological significance of such membrane acceptors.