A ROLE FOR THE INTERCHAIN DISULFIDE OR ITS PARTICIPATING THIOLS IN THE INTERNALIZATION OF BOTULINUM NEUROTOXIN-A REVEALED BY A TOXIN DERIVATIVE THAT BINDS TO ECTO-ACCEPTORS AND INHIBITS TRANSMITTER RELEASE INTRACELLULARLY

Botulinum neurotoxin type A consists of a disulfide-linked light and h eavy chain, with an intradisulfide present within the C-terminal half of the latter. The functional consequences of reducing these bonds and alkylating the thiols were investigated. Modification of free cystein e residues had no effect on the toxicity in mouse bioassays or on acet ylcholine release in the mouse nerve-diaphragm and the buccal ganglion of Aplysia californica. However, reduction of the toxin prior to alky lation drastically decreased neuroparalytic potency; yet, this derivat ive inhibited transmitter release if injected directly into a presynap tic neuron in the Aplysia ganglion or added to bovine permeabilized ad renal chromaffin cells. Its antagonism of the action of botulinum neur otoxin A at mammalian motor nerve endings and Aplysia neurons indicate s retention of the ability to bind to the toxin's productive ecto-acce ptors. Thus, the abolition of the toxicity of extracellularly applied botulinum neurotoxin A by the cleavage of both disulfides, and the alk ylation of the half-cystines involved, results from ineffective uptake . Modified forms of the isolated chains of botulinum neurotoxin A were utilized to determine which of the disulfides were necessary for inte rnalization. Alkylation of the cysteines in the light and heavy chains , including those involved in the interchain bond but excluding those of the intact disulfide in the heavy chain, revealed that the intermol ecular bond must be present, or the thiols concerned unmodified, for b otulinum neurotoxin A to undergo membrane translocation into Aplysia n eurons.