PROCESSING OF TETANUS AND BOTULINUM-A NEUROTOXINS IN ISOLATED CHROMAFFIN CELLS

Tetanus and botulinum A neurotoxins were introduced into the cytosol o f chromaffin cells by means of an electric field in which the plasma m embrane is forced to form pores of approximately 1 mu m at the sites f acing the electrodes. As demonstrated by electron microscopy, both [I- 125] and gold-labelled tetanus toxin (TeTx) diffuse through these tran sient openings. Dichain-TeTx, with its light chain linked to the heavy chain by means of a disulfide bond, causes the block of exocytosis to develop more slowly than does the purified light chain. The disulfide bonds, which in both toxins hold the subunits together, were cleaved by the intrinsic thioredoxin-reductase system. Single chain TeTx, in w hich the heavy and light chains are interconnected by an additional pe ptide bond, was far less effective than dichain-TeTx at blocking exocy tosis, which indicates that proteolysis is the rate-limiting step. The toxins were degraded further to low-molecular weight fragments which, together with intact toxins and subunits, were released by the cells. The intracellular half-life of [I-125] dichain-TeTx was approximately three days. The number of light-chain molecules required to maintain exocytosis block in a single cell, as calculated by two different meth ods, was less than 10. The long duration of tetanus poisoning may resu lt from the persistence of intracellular toxin due to a scarcity of fr ee cytosolic proteases. This may also hold for the slow recovery from botulism.