PREPARATION AND CHARACTERIZATION OF HOMOGENEOUS NEUROTOXIN TYPE-A FROM CLOSTRIDIUM-BOTULINUM - ITS INHIBITORY-ACTION ON NEURONAL RELEASE OF ACETYLCHOLINE IN THE ABSENCE AND PRESENCE OF BETA-BUNGAROTOXIN

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  • 1 January 1982
    • journal article
    • research article
    • Vol. 122  (3) , 493-500
Abstract
Large-scale production and purification of complexes between C. botulinum neurotoxin and hemagglutinin were achieved. Hemagglutinin-free neurotoxic protein of the complexes was purified to high specific neurotoxicity by affinity chromatography, on p-aminophenyl .beta.-D-thiogalactopyranoside coupled to Sepharose 4B, followed by chromatography on DEAE-Sephacel. The resultant neurotoxin was homogeneous on isoelectric focussing (isoelectric point = 6.3) and on dodecylsulfate polyacrylamide gel electrophoresis under non-reducing conditions when its MW was 1.4 .times. 105; after reduction, 2 polypeptides (MW = 9.9 and 5.5 .times. 104) were present. On double-immunodiffusion gels, using antiserum against neurotoxin-hemagglutinin complex, the neurotoxin showed a single, sharp precipitin line that was immunologically distinct from a relatively non-toxic protein (MW = 1.3 .times. 105), which co-purifies with the neurotoxin but is removed by the ion-exchange chromatography step. Application of the neurotoxin to animals in vitro or in vivo produced near complete and irreversible blockade of neurotransmission. Botulinization of rat leg muscles reduced spontaneous transmitter release; the amplitude of miniature end-plate potentials was altered from the normal bell-shaped to a skewed distribution. In normal muscle, a large transient increase in frequency of the miniatures was produced by .beta.-bungarotoxin. In contrast with botulinized muscle, the latter induced a much smaller increase in the absolute frequency; the mean amplitude was increased somewhat but the distribution remained skewed. Evidently, botulization of muscle modifies the action of .beta.-bungarotoxin.