Disulfide formation in reduced tetanus toxin by thioredoxin: the pharmacological role of interchain covalent and noncovalent bonds.
The interchain disulfide bond of tetanus toxin is known to be cleaved by reduced thioredoxin and by rat brain homogenate. We now show that this bond, but not the disulfide loop in the heavy chain of the toxin, can be restored quickly and completely by oxidized thioredoxin. Oxidized glutathione was at least 100 times less potent and less specific. Reduced tetanus toxin did not measurably (KD below 50 nM) dissociate into its chains, as revealed by HPLC gel chromatography under nondenaturing conditions. Accordingly, when the reduced toxin or its recombined chains were injected into mice, general toxicity was diminished but not abolished, as compared with the native form. Inhibition of Ca(2+)-evoked [3H]noradrenaline release was assayed in cultured adrenomedullary cells after permeabilization with digitonin. Reduced two-chain tetanus toxin was as active as the isolated light chain in this system, and the action of the light chain was only slightly diminished by the addition of excess heavy chain. The results show that thioredoxin can both open and close the covalent bond between the chains of tetanus toxin, and that the reduced chains remain linked by noncovalent forces. The role of the thioredoxin system for reversible activation of tetanus toxin in vivo remains to be established.
Kistner
A
A
Rudolf-Buchheim-Institut für Pharmakologie, Justus-Liebig Universität, Giessen, F.R.G.
Sanders
D
D
Habermann
E
E
D000313:Adrenal Medulla / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000378:metabolism* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D009638:Norepinephrine / Q000378:metabolism; D010084:Oxidation-Reduction; D013329:Structure-Activity Relationship; D013744:Tetanus Toxin / Q000378:metabolism* / Q000633:toxicity; D013879:Thioredoxins / Q000494:pharmacology*
D004220:Disulfides; D013744:Tetanus Toxin; D013879:Thioredoxins; D009638:Norepinephrine