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Neurotoxins in axenic oscillatorian cyanobacteria: coexistence of anatoxin-a and homoanatoxin-a determined by ligand-binding assay and GC/MS

Rómulo Aráoz^1,

Nicolae Palibroda^3,

¹ Unité des Cyanobactéries (CNRS URA 2172), Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris Cedex 15, France
² Unité des Récepteurs et Cognition (CNRS URA 2182), Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris Cedex 15, France
³ Laboratoire de Chimie Structurale des Macromolécules, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris Cedex 15, France

Correspondence
Michael Herdman
mherdman{at}pasteur.fr

Two neurotoxic alkaloids, anatoxin-a and its homologue homoanatoxin-a, were purified from the filamentous cyanobacteria Oscillatoria sp. strain 193 (PCC 9240) and Oscillatoria formosa NIVA CYA-92 (PCC 10111), respectively, and characterized by mass spectrometry. Biological activity was determined by examining the capacity of the toxins to competitively inhibit the binding of radiolabelled bungarotoxin to acetylcholine receptors, using post-synaptic membrane fractions of Torpedo electric tissue. Inhibition was concentration dependent, with a K_i of 5·4±1·1x10^–8 M for anatoxin-a and 7·4±0·9x10^–8 M for homoanatoxin-a. Their high affinities for the nicotinic cholinergic receptors were exploited to adapt the radioligand-binding assay for routine detection of this class of neurotoxins directly in low-molecular-mass cell extracts of cyanobacteria. Confirmation of the results and toxin identification were achieved by coupled gas chromatography-mass spectrometry (GC/MS). Seventy-six axenic strains, representative of 13 genera, were analysed. Five strains of the genus Oscillatoria, hitherto unknown for their toxicity, inhibited bungarotoxin binding. GC/MS revealed that Oscillatoria sp. strains PCC 6407, PCC 6412 and PCC 9107 synthesized exclusively anatoxin-a, whereas both anatoxin-a and homoanatoxin-a were produced by strain PCC 9029. Oscillatoria sp. strain PCC 6506, an isolate co-identic with strain PCC 9029, also produced both neurotoxins, but their respective presence depended upon growth conditions. The latter results suggest that regulatory differences in at least some of the cyanobacterial strains may account for the preferential synthesis of only one of the two neurotoxins or for their simultaneous occurrence.

Present address: Unité de Génétique Moléculaire Bactérienne, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France.

Present address: Institute of Isotopic and Molecular Technologies, 71-103 rue Donath, PO Box 700, RO-3400 Cluj-Napoca 5, Romania.

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