Fluoro AChE

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Introduction

Exposure to chemical nerve agents, pesticides and certain drugs (anesthetics, cocaine and therapeutical drugs) reduces the activity of red blood cell (RBC) acetylcholinesterase (AChE). The RBC-AChE can be used as a biomarker to monitor suppressed and or increased AChE function in the peripheral and central nervous system (9). Acetylcholinesterase (AChE) is one of the most important enzymes involved in nerve transmission. The enzyme is bound to cellular membranes of excitable tissue (synaptic junction, endoplasmic reticulum, etc) 1-3. Acute toxicity to humans and animals through inhibition of AChE by both nerve gases and an important class of pesticides has long been a field of intensive scientific investigation 4,5. AChE inhibitors have also been used clinically as Alzheimer’s treatments (e.g., tacrine (tetrahydroaminoacridine)) 6 and are the subject of increasing interest in various disease processes and treatment strategies 7,8. However, both environmental detection of AChE inhibitors and development of modulators of AChE enzymatic activity as drugs have been hampered by the difficulty and complexity of the current assay methods.

Key Benefits

  • Non Radioactive assay can monitor multiple time points to follow kinetics.
  • One-step, no wash assay.
  • Versatile: Can Detect AChE activity in RBC’s, Saliva and Tissue Lysates.
  • Readout – 96 well Fluorescent Plate Reader.

Additional information

Kit Size

100, 500

We have developed a highly sensitive, very rapid, extremely simple assay to determine acetylcholinesterase activity in RBC’s, using the natural substrate, acetylcholine. Additionally, by using specific inhibitors, the kit can be used to detect AChE activity in a variety of samples. A series of coupled enzyme reactions quickly translates the presence of active AChE into a change in the fluorescence of a quenched detection reagent.

AChE + ATP + H20 + coupled enzyme reaction + quenched dye —-> Fluorescent

Dye (Ex:530-570nm

Em:590-600nm

fluoro_ache_detection_in_rbc

Time=10 min Dilution Volume used ug protein/well RFU *
RBC 1:1000 10uL 5 3450
RBC no Ach 1:1000 10uL 5 152
Rat brain 1:500 10uL 20 5864
Rat brain no Ach 1:500 10uL 20 296
Saliva neat 10uL ND 925
Saliva no Ach neat 10uL ND 772

fluoro_ache

RBC-AChE mU/mL in well RFU SD RFU
167.000 9999 0.00
41.750 6375 121.70
10.438 1659 9.00
2.609 590 9.81
0.652 256 7.23
0.163 222 8.72
0 186 8.08
TitleFileLinkAuthor(s)JournalYear; Edition:Pages
Gonadal hormones modulate the potency of the disruptive effects of donepezil in male rats responding under a nonspatial operant learning and performance taskhttp://journals.lww.com/behaviouralpharm/Abstract/publishahead/Gonadal_hormones_modulate_the_potency_of_the.99913.aspxLeonard, Stuart T.; Hearn, John K.; Catling, Andrew D.; Winsauer, Peter JBehavioral PharmacologyFeb 2010
Acetylcholinesterase, a key prognostic predictor for hepatocellular carcinoma, suppresses cell growth and induces chemosensitizationhttp://onlinelibrary.wiley.com/doi/10.1002/hep.24079/fullYingjun Zhao, Xiaoying Wang, Tao Wang, Xin Hu, Xin Hui, Mingxia Yan, Qiang Gao, Taoyang Chen, Jinjun Li, Ming Yao, Dafang Wan, Jianren Gu, Jia Fan, Xianghuo HeHepatologyVol 53, Issue 2, pp 493–503, February 2011-DOI: 10.1002/hep.24079
Reference
Politoff, A., Blitz, A., and Rose, S.: Incorporation of Acetylcholinesterase Into Synaptic Vesicles is Associated with Blockade of Synaptic Transmission, Nature 256, 324, 1975
Friedenberg, R., and Seligman, A.: Acetylcholinesterase at the Myoneural Junction: Cytochemical Ultrastructure and Some Biochemical Considerations, J Histochem Cytochem 20, 771, 1972
Nachmansohn, D.: Proteins in Excitable Membranes, Science 168, 1059, 1970.
HA Berman and MM Decker Kinetic, equilibrium, and spectroscopic studies on dealkylation ("aging") of alkyl organophosphonylacetylcholinesterase. Electrostatic control of enzyme topography. J. Biol. Chem., Aug 1986; 261: 10646-10652 .
Arie Ordentlich et al. The Architecture of Human Acetylcholinesterase Active Center Probed by Interactions with Selected Organophosphate Inhibitors. J. Biol. Chem., May 1996; 271: 11953-11962.
Levy R. Tetrahydroaminoacridine and Alzheimer's disease. Lancet, 1987 Feb 7;1(8528):322.
Bolognesi ML et al. Propidium-based polyamine ligands as potent inhibitors of acetylcholinesterase and acetylcholinesterase-induced amyloid-beta aggregation. J Med Chem. 2005 Jan 13;48(1):24-7.
Schallreuter KU et al. Activation/deactivation of acetylcholinesterase by H2O2: more evidence for oxidative stress in vitiligo. Biochem Biophys Res Commun. 2004 Mar 5;315(2):502-8.
# Nigg HN, Knaak JB. Blood cholinesterases as human biomarkers of organophosphorus pesticide exposure. Rev. Environ. Contam. Toxicol, 2000:163: p29-111.
Part#ReagentTemperature
Part# 3023Component A: Detection Reagent Diluent, 5.5mL-20C
Part# 3024Component B: Coupled Enzyme Reagent, 5.5mL-20C
Part# 4016Component C: Detection Reagent, 1 Vial-20C
Part# 3011Component D: 5X Reaction Buffer-20C
Part# 7012Component E: Acetylcholine-20C
Part# 6020Component E: Reg Blood Cell Acetylcholinesterase-20C

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