Binding of QNB and Atropine to Muscarinic Acetylcholine

Binding of QNB and Atropine to Muscarinic AcetylcholineCholinergic relates to the responses in various systems to the neuro-transmitter molecule Acetycholine (ACh). They atomic number 18 the protein that atomic number 18 permanently attached to the biological membrane or the integral membrane protein (IMP). If the set of response is seen where Ach is a normal transmitter it is seen that they are grouped found on nicotinic acetylcholine sense organs (nAChR) that respond to nicotine, and muscarinic acetylcholine receptors (mAChR) that bind muscarine. These Nicotine and muscarine are extrinsic molecules that get the same response but with different sensitivity. Drugs that bind to muscarinic receptors are classified based onAgonists (which activate the neuronal receptor and produce a response)Antagonists (which do not activate the receptor and block the agonist binding site)Antagonists are now employ to study the drug-receptor binding as they bind with a higher kinship (i.e lower di ssociation constant kd) when compared with agonistsPharmacology studies have shown that antagonists have higher affinity but no efficacy to their cognate receptors. They intervene their effect by going and binding to the active site or to allosteric sites on the receptor. They can besides go and bind to unique binding sites that do not participate in biological regulation of any receptor activity. The activity that antagonist causes whitethorn be reversible or irreversible, depending on the long life of the antagonist-receptor complex.Studies have shown that 3-Quinuclinidyl benzilate (QNB) is a potent muscarinic antagonist in CNS (central nervous system) and peripheral tissues. QNB shows ad hoc binding to the receptor of interest it binds. It can in like manner bind to other sites of the membrane and these can cause changes. We can vizor specific binding by filtering radioactive 3H-QNB and then beat the amount of QNB. To measure non-specific binding, Atropine is used to displac e QNB from the specific sites, while the non-specifically adjoin QNB remains and can be quantified by measuring radioactivity.(Source Yamamura et al. May 1974)Overview of the experimentQNB is carried out in a radioactive binding assay where the preoccupancy of QNB that is specific bound without atropine and QNB that is non-specifically bound with atropine is measured over consequent interval of meter. It is allowed to incubate so as for binding site to reach saturation is allowed where the equilibrium is reached. After this any further increase incubation time does not cause the amount of QNB bound to change. This QNB bound to the membrane is measured. By designing the incubation time, IC50 of atropine is measured by measuring the atropine at which 50% of bound QNB is displaced. Amount of free QNB when 50% of bound QNB is displaced is used to measure the dissociation constant (Kd).Materials and MethodsDetermoation of QNB specific and non-specific bindingTwo bulk assays was car ried outTo measure QNB binding (in the presence of water)To measure non specific binding (with the presence of atropine)There were two conical flask taken A and B. Tube A was added with 30 ml of 1.3 nM 3H-QNB and 6ml water. And to the flask B flask B, 30 ml 3H-QNB and 6ml atropine was added. S filter tower is then set with 6 GF/C filters and 4.0 ml of rat membrane was added to each flask and the flask were swirled to mix well. 2ml aliquots from A flask (A1, A2, A3) and (B1, B2, B3) from the B flask were produced and were run through fresh GF/C filters. severally of the filters was then washed to remove mini-vials, and then 5 ml scintillant was added and was odd for at least an hour. After a hour the radioactivity was counted in the scintilliant counter. This protocol was repeated for a couple of more time to produce triplicates at the time interval of 10, 20, 30, 45 and 60 min.Determination of IC50 for atropineFive fruitcake test thermionic valves having 1200 l of distilled wate r in each was taken. To the test tube 1, ccc l of 10 10 M atropine was added and was mixed well. 300 l of the solution was added to tube 2 and mixed well. The same method is carried out for a series of dilutions to be done in tube 3 to 5. Atropine concentration in each tube is calculated.Seven triplicate tubes (A1, A2, A3G1, G2, G3) are make each containing 1500 l of 1.3nM QNB assay and the tubes are mixed well. 300 l of 10 M atropine was added to the three tubes of A and three B tubes were added with 300 l of solution from tube 1. The dilution process was carried out for tubes C, D, E, F from tube 2, tube 3, tube 4 and tube 5 respectively. To tubes G, 300 l of distilled water was added instead. 200 l membrane was then added quickly to all the tubes. The 21 tubes were then left for incubation for 45 min and the radioactivity was then measured.Determination of concentration of protein using Lowry AssayTest tubes were prepared that contained 0, 50, 100, 150 and 200 g BSA (Bovine bloo d serum albumin) made up to 1 ml with water. A 6th tube was taken that had 50 l of membrane that was made up to 1ml with water. 1.5ml of reagent 1 that contains 0.5 ml copper tartrate + 50ml alkaline carbonate was added and mixed well and let to stand for 10 min at room temperature. Then 0.3 ml of reagent 2 that contains moneymaking(prenominal) Folin-Ciocalteau reagent was added to the tubes and mixed well. The tubes were then left for incubation for 30 min. Absorbance or optical density was read at 660nm.Determination of kd for QNBEight test tube was taken, tetrad containing low QNB concentration (1.3nM QNB mix) and four tubes containing high QNB concentration (6.5nM QNB mix). Tubes 1 to 4 were added with 7.50 ml, 2.50 ml, 5 ml and 3.2 ml of 6.5 nM QNB mix respectively. Lower concentration of QNB is made by diluting the standard QNB assay mix with NaKP solution. These tubes are labelled 1-8. The solution of tube 1-8, of about 1500 l each was added to the triplicate tubes (A1, A2, A3, H1, H2, H3) respectively. Solution of tube 1 is added to tubes A, Tube 2 to B tubes till tube 8 to tubes H. 300 l water + 200 l membrane was then added to all tubes. For tubes A4-H4, 300 l Atropine plus (Tube 1-8) respectively plus 200 l membranes was added. Radioactivity was measured in all tube. A lowry assay was also carried out.RESULT AND reciprocationHere in the graph the values are plotted for QNB bound with atropine (with as show in the graph), QNB bound without atropine (Without as shown in the graph) and Corrected vales are obtained by subtracting QNB bound with atropine from the QNB bound without atropine (corrected as shown in the graph ) against time. Here QNB bound without atropine is total amount of QNB bound to the receptor QNB bound with atropine is the Non-specific binding of QNB to the receptor and corrected is the specific binding of QNB to the receptor.After a particular time of incubation receptors reach equilibrium, where no more binding of QNB takes plac e to the binding sites. At this point when no more binding of QNB takes place the plateau is formed in the graph showing saturation. This incubation time is slightly 45 min as shown by the graph reaching the plateau.The graph shows us that with and corrected points of the graph forms a plateau after reaching incubation time of approximately 45 min. If an addition incubation time was taken after 60 min we would have got a plateau for without graph also showing us a plateau.The graph shows that the cmp value increase over time after which when reaching a particular time no more binding occurs then forming a plateau showing the saturation or equilibrium has reached. Small decline in the graph can be seen at time 30 to 45 min, this could have been due to experimental errors. The errors could have been caused during pipetting, in proper vacuum, formation of bubbles, adding samples properly between time intervals etc. This can be avoided by more careful handling of the instrument and do ing a initial check up for errors so as to not cause changes in the experiments result.Taking the higher up data into consideration we have chosen 45 min as incubation time for determine IC50 of atropine. This is because, saturation of binding sites is achieved and no further unbinding of QNB also occurs, as the off-rate or reaction constant of QNB unbinding is very low. So there is no further change in the amount of bound QNB and hence this incubation time is considered appropriate.By serial dilution different concentration of atropine was prepared. The graph shows us that the amount to QNB bound to the receptor of the membrane reduces with increase in concentration. This happens because atropine is a agonistical binder and binds competitively with specific sites to the receptor. The amount of QNB specifically bound will be inversely proportional with atropine concentration.Half maximal inhibitory concentration (IC50)is a measure of how effective a compound is in inhibiting biolo gical or biochemical function. This is a quantitative measure that let us know how a frank deal concentration of the drug or biological substance (inhibitor) is required to inhibit a given biological process by half. So we are calculating the IC50 of atropine to determine its potency. It is calculated by taking atropine concentration at which 50% QNB is displaced. The IC 50 value was found to be 0.0008912 M. This shows that atropine is a drug with good potency. Ic 50 does not directly discuss the binding constant so we cannot compare the binding affinity of QNB and receptor.Lowarys assayLowrys assay was carried out for determining the concentration of membrane protein. First different concentration of BSA was used and we generated a graph for it, taking concentration and OD. The membrane protein was then checked for absorbance and was found to be 0.322. Using the linear regression equation and the absorbance, concentration of the membrane protein was found to be 0.803 mg/ml.This t est was done for another membrane protein sample. The absorbance of the membrane was 0.27. again using the regression equation and the absorbance, concentration of the membrane protein was found to be 0.293529412 mg/ml.Determination of KdKd is -1/m and was the equation was used is y = -8499.6x 1.3669. the kd is used to desexualize the affinity between the drug and the protein . the value of Bmax was 0.001161 nm.