Evidence for a link between specific proteolysis and inhibition of [3H]-noradrenaline release by the light chain of tetanus toxin.
The light chain of tetanus toxin is known to inhibit the Ca(2+)-evoked release of [3H]-noradrenaline from digitonin-permeabilized bovine adrenomedullary cells in culture but does not change the basal outflow or the total cellular radioactivity. Evidence for the involvement of proteolysis in this effect was obtained by three approaches. First, the permeabilized cells were exposed to a series of enzymes. The endoproteinase Glu-C mimicked the inhibition produced by the light chain. Second, protease inhibitors of different specificities were assessed for blockade of the action of light chain on [3H]-noradrenaline release from permeabilized cells. Blockade was complete with EDTA (2.5 mmol/l) or 1,10-o-phenanthroline (1 mmol/l), and absent with the highest concentrations tested of diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, pepstatin, leupeptin, bestatin, phosphoramidon, thiorphan or trans-epoxysuccinic acid (E64) which is regarded as an inhibitor of thiol proteases. This inhibitor spectrum suggested that light chain might be a metalloprotease. Finally a sequence-His-Glu-Leu-X-His-occurring in the light chains of tetanus toxin and of the botulinum neurotoxins A, C, D, E was also found in many endoproteinases and an aminopeptidase. The motif is known to constitute their active site and to bind Zn2+. In fact Zn2+ (0.6-0.9 mol/mol) was found in thoroughly dialysed two-chain tetanus toxin. The three approaches jointly support the hypothesis that the light chain of tetanus toxin, and probably of all clostridial neurotoxins, inhibits [3H]-noradrenaline release from adrenomedullary cells by degradation of (a) specific, still unknown protein(s) involved in exocytosis.
Sanders
D
D
Rudolf-Buchheim-Institut für Pharmakologie, Justus-Liebig-Universität Giessen, Federal Republic of Germany.
Habermann
E
E
D000313:Adrenal Medulla / Q000201:enzymology / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002838:Chromaffin System / Q000201:enzymology; D066298:In Vitro Techniques; D007525:Isoelectric Focusing; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009638:Norepinephrine / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D013744:Tetanus Toxin / Q000494:pharmacology*
D011480:Protease Inhibitors; D013744:Tetanus Toxin; D008666:Metalloendopeptidases; D002118:Calcium; D009638:Norepinephrine