Qnb And Atropine Binding To Muscarinic Acetylcholine Receptor Biology Essay

Qnb And Atropine Binding To Muscarinic Acetylcholine Receptor Biology EssayUsing rat brain membranes, buffer, atropine and 3H-QNB you will produce a displacement draw in for QNB by atropine, using a filtration method to separate marge QNB from free QNB. Radioactivity on the filters will be mea genuined by scintillation figuring and, after correction for counting efficiency, will be converted into molar units from special(prenominal) radioactivities.IntroductionReceptors for acetylcholine ar present in many tissues and can be characterised as f every(prenominal)ing into cardinal main types, muscarinic or nicotinic, on the basis of their ability to bind muscarine or nicotine respectively. Several substances are known that bind to the muscarinic acetylcholine sense organ (mAChR) some of these are agonists (which bind and elicit a response) and some are antagonists (which bind but do not elicit a response). In general, antagonists are used to measure receptor medical dressing as they bind with a steeper affinity (lower KD) than agonists bind.In this try you will investigate some of the properties of mAChR in rat brain membranes by means of 3H-quinuclidinyl benzilate (3H-QNB) binding.This experiment is based upon an article by Yamamura Snyder (1974) Proc Natl Acad Sci USA 71 1725-1729 (See course website.)Requirements1. Rat brain membranes store on ice. (See p for expression method).2. Sodium potassium phosphate (NaKP) 50 mM pH 7.4standard 3H-QNB/NaKP search blend (NaKP + 1.3nM 3H-QNB,11.2 x 102 Bq/pmolhigh concentration 3H-QNB/NaKP assay mix (NaKP + 6.5 nM 3H-QNB,11.2 x 102 Bq/pmolatropine solution (10 M MW 290)* QNB AND ATROPINE ARE TOXIC SO HANDLE WITH CARE *3. Small rubbish test tubes, micropipettes 200 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 scintillantMethodsAll assays have a final volume of 2.0 ml, ma de up of 1.5 ml 3H-QNB assay mix, 0.3 ml body of water or atropine. The assay is started by adding 0.2 ml membranes. The excess atropine added to the controls displaces the specific and saturable (i.e. receptor-bound) QNB expiration the non-specific, non-saturable QNB bound to the membranes.The assays are left for the appropriate length of time, stopped by adding 2.0 ml NaKP to increase the volume and filtering immediately through glass fibre filters. These are washed with NaKP and counted overnight in a scintillation counter.Day 11. Make up twain bulk assays, one to measure total QNB binding (with water) and one to measure non-specific binding (with atropine). Set up two 50 ml conical flasks 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 vi GF/C filters. When you are ready, quickly add 4.0 ml swirled membranes to each flask and swirl to mix.3. at present remove 2.0 ml al iquots to filters, three for each flask, making sure that you know which are from flask A and which from B.*USE SEPARATE PIPETTE TIPS FOR FLASKS A AND B*Note that if you contaminate the QNB solutions with atropineit will completely abolish all bindingFilter quickly through fresh 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 radioactivity in the scintillation counter.5. Repeat 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 example by rubbing gloves, bench or anything that top executive have been in contact with 3H-QNB. Carefully note the origin of each swab. Then put 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 standard safety procedure when dealing with radioactive chemicals. The amounts of tritium involved in this experiment are un probable to damage your health. Nevertheless this is a useful exercise to find test your technique before you make a mistake with 32P or 125I (much more than damaging).Day 2Note 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 meliorate 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 1200 l of distilled water in each.Now add 300 l of 10 M atropine to Tube 1, mix well and conveyance of title 300 l to Tube 2.Mix well and transfer 300 l to Tube 3.Repeat up to Tube 5.Calculate the atropine concentration in each tube.Set up 7 triplicate glass tubes (A1, A2, A3, B1 G3) as followsTubes300ml of1.3 nM QNB assay mixA10mM atropine1500mlBTube 11500mlCTube 21500mlDTube 31500mlETube 41500mlFTube 51500mlGdistilled water1500mlAs rapidly as possib le add 200ml membranes to each tube. Proceed as described in 2).4) above, using the incubation time you calculated from Day1s experiment (it should be at least 45 minutes). It is best to start the reactions in two batches, with 5 minutes between each batch to allow you time to filter the first batch before the second batch is due.Calculate the clean radioactivity bound to each triplicate set of filters and convert this value into suitable units of QNB bound (nanomoles or picomoles). Plot these values against log10atropine. Estimate the IC50 from the midpoint of the ignore and compare your result with that obtained by Yamamura Snyder.While you are waiting for the reactions to reach 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 given 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 give out 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. number the water or atropine last.Tubes1500 l from Tube 300 lA1 irrigateB2water systemC3WaterD4WaterE5WaterF6WaterG7WaterH8WaterNow label a separate set of eight tubes label A4, B4H4. Set these up as the previous but add Atropine instead of water. Note that this set is not done in triplicate.Add 200 l of membrane preparation to each tube. Incubate the tubes as describe d in 2)4) above, the incubation time being 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). Draw 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 data 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 analysis. This will be discussed during the following session. Further information about this and other methods of analysis can be found ath ttp//www.curvefit.com/introduction75.htm lock away of your radioactive equipment and toxic chemicals in the correct places.Data analysisQuestions to think aboutHow many dpm should be present in each assay? (Calculate this.)What is the likely nature 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 affect the IC50 of atropine?LOWRY ASSAY FOR PROTEINReagent 10.5 ml copper tartrate has been mixed with 50 ml saltlike 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. arrive at 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 room temperature.Add 0.3 ml Reagent 2, mix well and leave for 30 min.Read 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 sucrose/0.1 mM PMSF with a Teflon-glass Potter homogeniser. This was centrifuged at 12000g x 10 minutes and the gibe resuspended in original volume of sucrose and frozen in aliquots.(PMSF = phenylmethylsulphonylfluoride half-life in water c. 3hr)