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Cholinesterase prac notes
Key Concepts in Pharmacology (PCOL2021)
University of Sydney
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cCholinesterase’s and Inhibitors Practicals Info Aims and Background Aims There are two aims for the experiment:
- To qualitatively compare and contrast the substrate specificity of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in the presence of known and potential substrates.
- To qualitatively compare and contrast the sensitivity to inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) to known and potential cholinesterase inhibitors. Background Acetylcholine is synthesised from acetyl CoA and choline via choline acetyltransferase, as shown in figure 1. Coenzyme A and water are byproducts of this synthesis. Acetylcholine (ACh) is inactivated by a two-step hydrolysis to choline and acetate by the enzyme acetylcholinesterase (AChE). Acetylcholine is then metabolised by a two-step hydrolysis to choline and acetate by the enzyme acetylcholinesterase (AChE) in the extracellular environment, e. in the synapse or neuromuscular junction. It is this reaction that forms the focus of our practical Cholinesterases and Inhibitors. Figure 1: Diagram of acetylcholine synthesis and degradation. In humans and animals there are two major types of enzymes that hydrolyse acetylcholine (ACh) to choline and acetic acid, namely acetylcholinesterase (AChE) (figure 2) and butyrylcholinesterase (BuChE) (figure 3):
AChE, also known as true cholinesterase, is found in the peripheral and central nervous system at cholinergic synapses and neuromuscular junctions where its role is to hydrolyse (inactivate) ACh within cholinergic synapses. It is also found in the membranes of red blood cells. BuChE, also know as pseudocholinesterase, plasma or serum cholinesterase, is found in plasma and other tissues such as liver and muscle; its function is largely unknown. The two enzymes differ with respect to their substrate specificity. AChE has a narrow substrate specificity whereas BuChE has a relatively broad substrate specificity. BuChE hydrolyses butyrylcholine (BuCh) more rapidly than ACh, and hydrolyses other esters such as procaine and suxamethonium. The measurement of plasma BChE activity has been recommended for predicting the outcome from organophosphorous pesticide poisoning, however, the usefulness of this approach has been questioned (Eddleston et al., 2008). AChE and BuChE differ in their sensitivity to inhibitors (anticholinesterases). Inhibitors are traditionally divided into two groups: reversible and irreversible inhibitors. These inhibitors can be used clinically to increase the tone and motility of the intestine and bladder in atonic states, to constrict the pupil, reduce intra-ocular pressure in glaucoma, and to improve skeletal muscle function in myasthenia gravis. Organophosphorous inhibitors are still used as pesticides. The self-poisoning with organophosphorous insecticides is a major global health problem (Eddleston et al., 2008). Cholinesterases activity can be measured using a variety of approaches utilising colorimetric, fluorometric, radiometric and electrochemical techniques (Miao et al., 2010). Figure 1. Hydrolysis of acetylcholine to choline and acetic acid by acetylcholinesterase Figure 2. Hydrolysis of butyrylcholine to choline and acetic acid by butyrylcholinesterase.
Carbachol – a little hydrolysis Nitrogen instead of carbon – residence structure – incredibly slow Inhibition because it’s a slow reaction – get into catalytic sites meaning ach cant be hydrolysed Methacholine – AchE only Methyl group – steric hinderance – to big and bulky Suxamethonium – size didn’t hydrolyse BCHE slow
Edrophonium – inhibitor Only anchors to anionic site at glutamic acid
about 100 × Km to achieve near-maximum velocity (~99% saturation). The relationship between reaction rate and substrate concentration is described by the Michaelis–Menten equation, where: v = velocity of reaction Vmax = maximum rate achieved by the system [S] =concentration of a substrate S KM =Michaelis constant Figure 4: The effect of the substrate concentration on the rate of reaction.
Malathion is a long lasting (irreversible) AChE inhibitor. It is converted to the active metabolite in vivo and is used as a pesticide. This organophosphate is metabolised in higher animals (e. man and bird) by ester hydrolysis to less toxic derivatives. However, insects metabolise the P = S to a P = O group, generating the lethal form of the insecticide. Experimental Background The measurement of cholinesterases activity in this experiment is a simple colorimetric assay based on the fact that acid is liberated by the hydrolysis of acetylcholine. The degree of activity is quantitatively measured from the photometric change of phenol red (in Tris buffer) to yellow by spectrophotometric absorption at 557 nm. A decrease in the red colour (alkaline) of the indicator phenol red reflects (stoichiometrically) a decrease in alkalinity of the buffer due to the enzymatic liberation of acid. A solution of phenol red will have a yellow colour at a pH of 6 or below and a red colour at a pH of 8 and above. The change in colour of phenol from red to yellow colour can be visually monitored when the pH value decreases (Fig. 5). Figure 5. Example of how pH has an effect on the colour of phenol red; red indicates basic conditions while yellow indicates acidic conditions. Each group will investigate AChE (true) and BuChE (pseudo or serum). A preparation of AChE derived from eel red blood cells will be available. A preparation of BuChE from horse serum will also be available. There may be differences in specificity between this horse serum cholinesterase and human serum cholinesterase. IDENTIFY the following stock solutions and equipment at your workstation (see Fig. 6): 1. Known and potential substrates (acetylcholine, butyrylcholine, carbachol, methacholine and suxamethonium) 2. Known and potential inhibitors (physostigmine, neostigmine, edrophonium, malathion, atropine and carbachol) 3. Distilled water 4. Tris Buffer (without phenol red indicator) 5. Cholinesterase enzymes (AChE and BuChE) 6. 96-well microtitre plate x 2; you will just be using rows A & B (for AChE) and G & H (for BChE) o plate 1 you will be adding phenol red ± substrates or tris buffer (negative control) o plate 2 you will be adding your enzyme ± inhibitors (rows A and G) or water (rows B and H) 7. Single-channel pipette and tips
Figure 6a. Stock solutions and equipment available at your workstation; ensure both 96- well plates are orientated with the letter A on the top left. Note: your plate that will eventually have phenol red and substrate will have a black line on the top.
Figure 7. Multichannel pipette Figure 8. Microplate reader Drugs to be tested Known Substrates Acetylcholine perchlorate 100 mM Butyrylcholine chloride 100 mM Potential Substrates Carbachol chloride 100 mM Methacholine chloride 100 mM Suxamethonium chloride 100 mM
Potential Inhibitors Physostigmine salicylate 5 μM Neostigmine bromide 5 μM Edrophonium chloride 80 μM Malathion 50 μM Atropine sulphate 5 μM Carbachol chloride 10 mM Enzymes Acetylcholinesterase (eel) Butyrylcholinesterase (horse) ** AChE and BuChE are diluted so that the rate of hydrolysis of the substrates ACh and BuCh, by the two enzymes, is equivalent Tris buffer indicator solution pH ~ 7.
Figure 11. Action of anticholinesterase drugs From: Rang HP, Ritter JM, Flower RJ, Henderson, G (2016) Rang & Dales's Pharmacology. Elsevier, Churchill Livingstone Fig. 13 p173. Note the following: R group length attached to ester group (green line), ester (red) and ionised amine (quaternary amines; blue)
Note the functional groups of the molecules above, in particular the amine attached to the ester for carbachol and the methyl group on the backbone of methacholine. Suxamethonium is just two acetylcholine molecules butted together.
Figure 11. Action of anticholinesterase drugs From: Rang HP, Ritter JM, Flower RJ, Henderson, G (2016) Rang & Dales's Pharmacology. Elsevier, Churchill Livingstone Fig. 13 p173. Methods Parts 1 and 2: Known and potential substrates and buffer (without indicator); and enzymes and inhibitors
Click to download template for substrates: 96-well plate and template that will be used to add substrate + phenol red
Cholinesterase prac notes
Course: Key Concepts in Pharmacology (PCOL2021)
University: University of Sydney
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