AChE

Metabolism / Metabolites
Anatoxin-a and homoanatoxin-a are potent neurotoxins produced by cyanobacteria such as Oscillatoria PCC 6506. Sequencing of the genome of this strain is underway, and ... a 29 kb DNA fragment containing a sequence called ks2 t... previously shown to be specific to Oscillatoria cyanobacteria producing anatoxin-a and homoanatoxin-a /was identified/. Bioinformatic analysis of this 29 kb fragment revealed a cluster of genes, which were annotated. The function assigned to the products of eight contiguous genes, from anaA to anaH, provides a clue to the biosynthesis of anatoxin-a and homoanatoxin-a. Proline is first loaded on an acyl carrier protein and its five-membered cycle oxidized to the pyrroline oxidation state. This activated ring is then successively loaded on three polyketide synthase modules for elongation, reduction, cyclization, and methylation. The final step is the hydrolysis of the thioester with subsequent decarboxylation. GC-MS and NMR analyses of homoanatoxin-a produced by PCC 6506 using labeled precursors confirm that proline is very likely the starter of these polyketide synthases. ... Specific PCR amplifications ... showed that the anaC, anaE, anaF, and anaG genes are always present in the genome of cyanobacteria producing anatoxin-a and homoanatoxin-a and absent in nonproducing strains. Histidine-tagged AnaC was purified to homogeneity and showed to catalyze the loading of proline on purified histidine-tagged AnaD that had been previously transformed into its holo form using the Bacillus subtilis Sfp phosphopantetheinyl transferase. All of these data provide strong evidence that ... the gene cluster responsible for the production of anatoxin-a and homoanatoxin-a in Oscillatoria PCC 6506 /has been identified/.

[1].An exploration of the binding prediction of anatoxin-a and atropine to acetylcholinesterase enzyme using multi-level computer simulations. Phys Biol. 2023 Nov 23;21(1).

[2].Anatoxin-a: Overview on a harmful cyanobacterial neurotoxin from the environmental scale to the molecular target. Environ Res. 2021 Feb;193:110590.

1-[(1r,6r)-9-Azabicyclo[4.2.1]non-2-En-2-Yl]ethanone has been reported in Oscillatoria, Microcystis aeruginosa, and other organisms with data available.

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Mechanism of Action
Anatoxin-a is a nicotinic (cholinergic) agonist that binds to neuronal nicotinic acetylcholine receptors. It has been suggested that the activation of presynaptic nicotinic acetylcholine receptors by anatoxin-a results in an influx of Na+, producing sufficient local depolarization to open voltage sensitive Ca++ and Na+ channels. The latter may then amplify the response, activating further Ca++ channels. As a result of this depolarization there is a block of further electrical transmission, and at sufficiently high doses this can lead to paralysis, asphyxiation and death. Anatoxin-a is more potent than nicotine or acetylcholine in evoking type 1A or type 2 current responses in rat hippocampal neurones, and it is more potent than nicotine in its ability to evoke the secretion of endogenous catecholamines from bovine adrenal chromaffin cells through their neuronal-type nicotinic receptors. Similar to nicotine, anatoxin-a was more potent than noradrenaline in releasing dopamine from striatal nerve terminals from rat superfused hippocampal synaptosomes. In vivo studies in the rat showed that the toxin stimulates the sympathetic system through the release of catecholamines from nerve endings.
Anatoxin-a is a nicotinic (cholinergic) agonist that binds to neuronal nicotinic acetylcholine receptors. It has been suggested that the activation of presynaptic nicotinic acetylcholine receptors by anatoxin-a results in an influx of Na+, producing sufficient local depolarisation to open voltage sensitive Ca++ and Na+ channels. The latter may then amplify the response, activating further Ca++ channels. As a result of this depolarization there is a block of further electrical transmission, and at sufficiently high doses this can lead to paralysis, asphyxiation and death.
Anatoxin-a (AnTx) is a natural neurotoxin, which acts as a potent and stereoselective agonist at the nicotinic acetylcholine receptors. ... The aim of this study was to determine the neurochemical bases for AnTx-induced striatal DA release, using the brain microdialysis technique, in freely moving rats. Local application of AnTx (3.5 mM) through the microdialysis probe produced an increase in striatal DA levels (701 +/- 51% with respect to basal values). The effect of infusion of AnTx in Ca(2+)-free Ringer medium, in Na(+)-free Ringer medium and with TTX in the medium, was inhibited. Also, reserpine pre-treatment blocked the action of AnTx on striatal DA levels. To investigate the involvement of the DA transporter, the effects of AnTx were observed in the presence of nomifensine. The coadministration of AnTx and nomifensine evoked an additive effect on striatal DA levels. The latter results show that the DA release is not mediated by a decreased DA uptake. Taken as a whole, these results suggest that the effects of AnTx are predominantly mediated by an exocytotic mechanism, Ca(2+)-, Na(+)- and TTX-dependent, and not by a mechanism mediated by the DA transporter.
(+)-Anatoxin-a (ANTX) stimulated guinea pig ileum contraction with a potency similar to that of acetylcholine (ACh); the stimulation was blocked by tubocurarine, hexamethonium, or atropine. Although the contraction stimulated by ANTX was blocked by atropine, no specific inhibition of the binding of [3H]N-methylscopolamine to ileum membranes was observed in the presence of ANTX. Furthermore, ANTX failed to stimulate the secretion of alpha-amylase from pancreatic acinar cells, a process that is activated by cholinergic agonists at the muscarinic receptors. When the ileum itself was stimulated by ACh, the contraction was not blocked by either hexamethonium or tubocurarine. Preincubation of the ileum with hemicholinium caused a 50% reduction in the ability of ANTX to stimulate contraction. Based upon these data, it was inferred that ANTX binds to postganglionic synaptic nicotinic receptors in the ileum, thus releasing endogenous ACh, which in turn causes ileum contraction by interacting with the postsynaptic muscarinic receptors. It was also observed that thymopentin (TP-5), a pentapeptide corresponding to positions 32-36 of thymopoietin, blocked the stimulation of ileum contraction by ANTX.

C10H15NO

165.23

165.115

64285-06-9

3034748

Oil

1.037 g/cm3

291ºC at 760 mmHg

124.7ºC

1.503

1.745

1

2

1

12

232

2

CC(=O)C1=CCC[C@@H]2CC[C@H]1N2

SGNXVBOIDPPRJJ-PSASIEDQSA-N

InChI=1S/C10H15NO/c1-7(12)9-4-2-3-8-5-6-10(9)11-8/h4,8,10-11H,2-3,5-6H2,1H3/t8-,10-/m1/s1

1-[(1R,6R)-9-azabicyclo[4.2.1]non-2-en-2-yl]ethanone

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Typically soluble in DMSO (e.g. 10 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)