The tetanus toxin light chain inhibits exocytosis.

The intracellular action on exocytosis of various forms of tetanus toxin was studied using adrenal medullary chromaffin cells, the membrane barrier of which has been removed by permeabilization with streptolysin O. Such cells still release catecholamines on stimulation with calcium. The two-chain form of tetanus toxin (67 nmol/l) strongly inhibited exocytosis, but only if dithiothreitol was present as a reducing agent. Purified light chain completely prevented [3H]noradrenaline release with a half-maximal effect at about 5 nmol/l. Heavy chain (up to 11 nmol/l) and unprocessed single-chain toxin (up to 133 nmol/l) were without effect. It is concluded that the original single-chain form of tetanus toxin has to be processed by proteolysis and reduction to yield a light chain which inhibits transmitter release.

Cooperative action of the light chain of tetanus toxin and the heavy chain of botulinum toxin type A on the transmitter release of mammalian motor endplates.

Purified heavy chain of botulinum toxin type A and light chain of tetanus toxin were combined to form a chimeric toxin. It was active on the mouse phrenic nerve-hemidiaphragm with a potency 6 times higher than that of native tetanus toxin. Electrophysiological data from poisoned neuromuscular junctions revealed that the pattern of nerve-evoked and spontaneous transmitter release was equivalent to that seen with tetanus toxin i.e. asynchronous release, and did not resemble that after botulinum toxin type A poisoning. We conclude that the light chain of tetanus toxin alone is responsible for the characteristic effects on spontaneous and nerve-evoked transmitter release of the native toxin and that these properties can be introduced into a new, more potent complex with the heavy chain of botulinum toxin A.

Palytoxin promotes potassium outflow from erythrocytes, HeLa and bovine adrenomedullary cells through its interaction with Na+, K+ -ATPase.

Erythrocytes from four mammalian species were compared with regard to K+ loss triggered by palytoxin, to Na+, K+ -ATPase activity, and to ouabain sensitivity of both events. Palytoxin sensitivity (EC50) decreased in the order rat, man (approximately equal to 1 pM) greater than cattle (approximately equal to 500 pM) greater than dog (greater than 10 nM). Na+, K+ -ATPase activity, as measured by Rb uptake, was in the series rat greater than man greater than cattle greater than dog. The glycoside potently inhibited both palytoxin action and ATPase activity in man, cattle and dog erythrocytes, but weakly in those from rats. Ca2+ promoted the palytoxin effects on all erythrocytes. As shown for human erythrocytes, Sr2+ and Ba2+ but not Mg2+ can substitute for Ca2+, and sucrose can substitute for sodium chloride. Human HeLa and bovine adrenomedullary cells also lost their K+ within a few min when exposed to palytoxin (1-10 pM). Ouabain acted as a palytoxin antagonist on both cell types. We conclude that: (a) the ouabain binding site of Na+, K+ -ATPase is part of the palytoxin receptor in every cell type tested, (b) high palytoxin sensitivity is not necessarily accompanied by high ouabain sensitivity, and (c) active ion transport is not a precondition for the action of palytoxin or for its inhibition by ouabain.

Increase of permeability of synaptosomes and liposomes by the heavy chain of tetanus toxin.

In search of a role for the heavy chain of tetanus toxin in poisoning, its actions on natural and artificial membranes have been assessed. The heavy chain increases the permeability of synaptosomes to lactate dehydrogenase and potassium ions, and promotes the outward shift of the lipophilic cation tetraphenylphosphonium which is a particularly sensitive indicator for depolarization. Independent of the assay system the potency of the heavy chain is high, i.e. in the range of about 1 nM, whereas its efficacy is low. Its potency is decreased by the addition of the light chain and by treatment of the synaptosomes with the C-terminal fragment C of the heavy chain, but not with its N-terminal fragment beta 2. Single- or two-chain toxin itself is inactive, and so are the light chain or the two heavy chain fragments beta 2 and C. Liposomes were made from phosphatidylcholine and phosphatidylserine or gangliosides and loaded with calcein. At pH 6 the outflow of calcein is promoted in the order heavy chain greater than toxin much greater than fragment beta 2, and the action of toxin is promoted by ganglioside. At pH 5, fragment beta 2 is nearly as active as the heavy chain and more potent than the toxin. The heavy chain, but neither of the fragments, is strongly adsorbed in hydrophobic interaction chromatography and caused aggregation of polystyrene-divinylbenzene beads. Evidence for polymerization of heavy chains is lacking in zonal centrifugation. It is concluded that both domains of the heavy chain co-operate to exert the membranal events described, and that the heavy chain is partially hidden by the light chain in the complete toxin molecule.

Chains and fragments of tetanus toxin. Separation, reassociation and pharmacological properties.

Tetanus toxin, as obtained from bacterial culture filtrates, consists of two chains. Since their roles in poisoning are unknown, we have made a detailed study of their preparation, reassociation and pharmacological activity. 1. Two-chain tetanus toxin (pI 6.0) was subjected to isoelectric focussing under reducing conditions in 2M urea. Both light (pI 4.8) and heavy (pI 7.2) chains separated as nearly homogeneous proteins of low toxicities. Upon removal of urea and reoxidation, partial homodimerization by formation of disulfide bonds took place in the purified fractions. The toxin was reconstituted nearly quantitatively by covalent heterodimerization of the complementary chains, as shown by SDS/gel electrophoresis, toxicity studies, inhibition of evoked [3H]noradrenaline release and binding to rat brain membranes. 2. Accordingly, fragment B (pI 5.6) resulting from papain hydrolysis, was separated into a light chain and the N-terminal moiety of the heavy chain, called fragment beta 2 (pI 7.1 and 6.8, two maxima). Removal of urea and reoxidation led to reconstitution of fragment B. Covalent linkage did not occur between the two parts of the heavy chain, or between the light chain and the C-terminal part of the heavy chain. 3. The heavy chain alone inhibited K+-evoked [3H]noradrenaline release from a rat brain homogenate. However, the concentration-response ratio was flat and 10-100-fold higher concentrations were required than with native or reconstituted two-chain toxin. The light chain was inactive. Purified heavy chain but not light chain decreased the [3H]noradrenaline content, whereas the two-chain toxin increased it. Binding to rat brain membranes was assessed by competition with 125I-labelled two-chain toxin. In hypotonic buffer, the heavy chain, the papain fragment C and native and reconstituted two-chain toxin had comparable affinities to membranes. In isotonic buffer the heavy chain displayed an about 1000-fold lower affinity than native or reconstituted two-chain toxin. The light chain did not bind to membranes in either test. Our data indicate that (a) the light chain and the N-terminal part of the heavy chain are held together not only by one disulfide bond but also by hydrogen bonds and ionic forces to yield a two-chain toxin or fragment B and (b) both chains contribute to the actions of the toxin in vivo and in vitro, and to its binding.

Chains and fragments of tetanus toxin, and their contribution to toxicity.

1. Single-chain toxin is enzymatically converted into two-chain isotoxins which differ from the precursor by their higher pharmacological activity, acidity and hydrophilicity. The interchain disulfide bridge and the disulfide loop within fragment C have been located at the amino acid level. 2. Independent of the enzymes used, the nicking sites are positioned within a region spanning no more than 17 amino acids. The N- and C-termini of the primary gene product are preserved in the two-chain toxin. The chains have been separated by isoelectric focussing and can be reconstituted to functionally intact toxin. 3. Light chain inhibits neurotransmitter release on different systems. First, permeabilized bovine adrenal chromaffin cells and rat pheochromocytoma (PC 12) cells release catecholamines when exposed to micromolar [Ca2+]. Inhibition is achieved with light chain or reduced two-chain toxin, but not with single-chain toxin or heavy chain. Washing away the light chain does not restitute the Ca2(+)-evoked release. The light chains of tetanus and botulinum A toxin act in a apparently similar, however not identical manner. Second, light but not heavy chain inhibits the release of acetylcholine when injected into Aplysia neurones. 4. The pharmacology of heavy chain is quite different. Ganglioside binding is mediated by its fragment C moiety, and modulated by the adjoining beta 2 piece and by light chain. Heavy chain and to a lesser degree its N-terminal beta 2-fragment promote the loss of calcein from liposomes indicating pore formation. Its C-terminal fragment C is inactive in this respect.(ABSTRACT TRUNCATED AT 250 WORDS)