Qnb And Atropine Binding To Muscarinic Acetylcholine Receptor Biology Essay

Qnb And Atropine Binding To Muscarinic Acetylcholine Receptor Biology Essay development rat point membranes, buffer, atropine and 3H-QNB you exit produce a displacement arch for QNB by atropine, using a filtration method to fork bound QNB from abandon QNB. Radioactivity on the filters go away be heartbeatd by flame counting and, after constituteion for counting efficiency, will be reborn into molar units from specific radioactivities.IntroductionReceptors for acetylcholine atomic number 18 present in umteen tissues and can be characterised as falling into 2 main types, muscarinic or nicotinic, on the basis of their ability to bind muscarine or nicotine respectively. Several substances ar known that bind to the muscarinic acetylcholine sensory receptor (mAChR) some of these are agonists (which bind and enhance a response) and some are antagonists (which bind but do not elicit a response). In general, antagonists are used to measure receptor binding as they bind with a higher(prenominal)er affinity (lower KD) than agonists bind.In this sample you will investigate some of the properties of mAChR in rat brain membranes by means of 3H-quinuclidinyl benzilate (3H-QNB) binding.This experimentation is based upon an article by Yamamura Snyder (1974) Proc Natl Acad Sci ground forces 71 1725-1729 (See course website.)Requirements1. Rat brain membranes store on ice. (See p for preparation method).2. Sodium potassium phosphate (NaKP) 50 mM pH 7.4 beat 3H-QNB/NaKP arrest mix (NaKP + 1.3nM 3H-QNB,11.2 x 102 Bq/pmolhigh concentration 3H-QNB/NaKP chit mix (NaKP + 6.5 nM 3H-QNB,11.2 x 102 Bq/pmolatropine solution (10 M MW 290)* QNB AND ATROPINE ARE venomous SO HANDLE WITH CARE *3. Small glass test tubes, micropipettes both hundred l (YELLOW TIPS), 1000 l (BLUE TIPS), 5000 l (WHITE TIPS)4. Multiplex filtration apparatus + GF/C glass fibre filters (2.5 cm diam) + forceps5. Scintillation mini-vials + Ultima Gold scintillantMethods whole assays have a fi nal volume of 2.0 ml, made up of 1.5 ml 3H-QNB assay mix, 0.3 ml water or atropine. The assay is started by fiting 0.2 ml membranes. The excess atropine added to the controls displaces the specific and saturable (i.e. receptor-bound) QNB leaving the non-specific, non-saturable QNB bound to the membranes.The assays are left for the becharm length of time, stopped by adding 2.0 ml NaKP to increase the volume and filtering straight off through glass fibre filters. These are washed with NaKP and counted overnight in a scintillation counter.Day 11. Make up two bulk assays, one to measure total QNB binding (with water) and one to measure non-specific binding (with atropine). Set up two 50 ml conelike flaskfuls thusAB3H-QNB (1.3 nM)30.0 ml30.0 mlwater6.0 ml0.0 mlatropine0.0 ml6.0 ml(this is enough for 20 assays you will do 18 assays)2. Set up a filter tower with sixsome GF/C filters. When you are ready, quickly add 4.0 ml swirled membranes to individually flask and swirl to mix.3. N ow obliterate 2.0 ml aliquots to filters, three for for for each one one flask, fashioning sure that you know which are from flask A and which from B.*USE start out PIPETTE TIPS FOR FLASKS A AND B*Note that if you contaminate the QNB solutions with atropineit will in all abolish all bindingFilter quickly through mellisonant GF/C filters.4. Wash each filter with 5 ml NaKP, remove filters to mini-vials, add 5 ml scintillant, invert, leave at least 1 hr, invert again and count the radiation in the scintillation counter.5. fictionalise steps 3 4 at times =10, 20, 30, 45 and 60 mins.6. Using the swabs provided, take six separate samples to check for radioactive contamination, for type scarters case by rubbing gloves, bench or anything that might have been in contact with 3H-QNB. Carefully note the origin of each swab. Then set apart each swab into a separate vial containing 5 ml of scintillant, as before, record the treatment of each, and send them for counting. This is a sta ndard pencil eraser procedure when dealing with radioactive chemicals. The amounts of tritium involved in this experiment are unlikely to damage your health. Nevertheless this is a useful exercise to feel test your technique before you curb a mistake with 32P or 125I (much much damaging).Day 2Note that you need to take capital care to get the correct volumes of each solution into the appropriate tubes. The more care you take, the better will be your resultsDetermine IC50 for atropine (i.e. that atropine concentration which displaces 50% of QNB binding).Take 5 small glass test tubes (1-5) and put 1 two hundred l of distilled water in each.Now add 300 l of 10 M atropine to underground 1, mix well and transfer 300 l to subway 2. flux well and transfer 300 l to Tube 3.Repeat up to Tube 5.Calculate the atropine concentration in each tube.Set up 7 reproduce glass tubes (A1, A2, A3, B1 G3) as followsTubes300ml of1.3 nM QNB assay mixA10mM atropine1500mlBTube 11500mlCTube 21500mlDTu be 31500mlETube 41500mlFTube 51500mlGdistilled water1500mlAs rapidly as possible add 200ml membranes to each tube. Proceed as described in 2).4) above, using the incubation time you pictured from Day1s experiment (it should be at least 45 transactions). It is best to start the reactions in two claspes, with 5 minutes between each batch to lease you time to filter the scratch batch before the second batch is due.Calculate the average radioactivity bound to each triplicate set of filters and convert this value into suitable units of QNB bound (nanomoles or picomoles). Plot these set against log10atropine. Estimate the IC50 from the midpoint of the curve and compare your result with that obtained by Yamamura Snyder. term you are waiting for the reactions to create equilibrium, carry out a Lowry assay for protein (see p) so that you can calculate specific QNB binding in fmol QNB per mg protein, and compare your value to that wedded in the Yamamura Snyder paper.You will be told in the class what quantities of membrane preparation to use in this assay.Day 3Note that you need to take great care to get the correct volumes of each solution into the appropriate tubes. The more care you take, the better will be your resultsDetermine KD for QNB. You will make lower concentrations of QNB by diluting the standard QNB assay mix with NaKP higher concentrations can be made from the high concentration 3H-QNB mix but this is strictly limited at 20 assays per group dont waste it.Label eight test tubes 1-8.Tube1.3 nM QNB mix6.5 nM QNB mixNaKPmlmlMl107.500.00202.505.00305.002.50403.204.3056.000.000.0062.500.005.0075.000.002.5083.500.004.00Label eight sets of triplicate tubes A1, A2, A3.H3. Add the water or atropine last.Tubes1500 l from Tube 300 lA1 urineB2WaterC3WaterD4WaterE5WaterF6WaterG7WaterH8WaterNow label a separate set of eight tubes label A4, B4H4. Set these up as the anterior but add Atropine instead of water. Note that this set is not make in triplicate.Add 200 l of membrane preparation to each tube. Incubate the tubes as described in 2)4) above, the incubation time be that determined on Day 1. It is best to start the reactions in two batches with 5 minutes between to allow you time to filter the first batch before the second batch is due.Calculate the average radioactivity bound to each triplicate set of filters and convert it into amounts of QNB (nano- or picomoles). move out a straight line through the atropine controls, and subtract the values for each real or estimated atropine control from the water values and use these info to calculate the bound and free QNB values.While you are waiting for the reactions to reach equilibrium, carry out a Lowry assay for protein (p) so that you can calculate specific QNB binding in fmol QNB per mg protein, and compare your value to that given in the Yamamura Snyder paper.The data from this experiment may be analysed by Scatchard psychoanalysis. This will be discussed during the following se ssion. Further information about this and otherwise methods of analysis can be found athttp//www.curvefit.com/introduction75.htmDispose of your radioactive equipment and poisonous chemicals in the correct places.Data analysisQuestions to think aboutHow numerous dpm should be present in each assay? (Calculate this.)What is the likely temper of the non-specific binding?Comment on the rate of binding for the specific and the non-specific binding.What other methods are available for measuring receptor-ligand equilibria?If the off-rate were fast (e.g. half-life of around 1 second) what method of assaying the receptor-ligand binding might be suitable?Does the QNB concentration dissemble the IC50 of atropine?LOWRY ASSAY FOR PROTEINReagent 10.5 ml copper tartrate has been mixed with 50 ml alkaline carbonate on the day of use.copper tartrate (0.1 g CuSO4.5H2O added to 0.2 g NaK tartrate in 20 ml water)alkaline carbonate (2 g NaOH in 20 ml water and adding 10 g Na2CO3, made up to 100 ml with water)Reagent 2Commercial Folin-Ciocalteau reagent 11 in waterMethodIn a series of test tubes, add the volume of membrane announced at the start of the class and make this up to 1 ml with water.Prepare tubes containing 0, 50, 100 150 and 200 g bovine serum albumin (BSA) made up to 1 ml water. The concentration of BSA you are supplied with is 1 mg.ml-1.Add 1.5 ml Reagent 1.Mix well and leave to stand for 10 min at elbow room temperature.Add 0.3 ml Reagent 2, mix well and leave for 30 min. take in at 660 nm.Plot the data from the standard BSA tubes and calculate the protein concentration in the membranes.PREPARATION OF RAT BRAIN TISSUESRat brain membranes for QNB binding experimentRat brains were homogenised in 10 volumes ice-cold 0.32 M saccharose/0.1 mM PMSF with a Teflon-glass Potter homogeniser. This was centrifuged at 12000g x 10 minutes and the pellet resuspended in original volume of sucrose and frozen in aliquots.(PMSF = phenylmethylsulphonylfluoride half-life in water c. 3hr)