Qnb And Atropine Binding To Muscarinic Acetylcholine Receptor Biology Essay

Using rat encephalon membranes, buffer, atropine and 3H-QNB you will bring forth a supplanting curve for QNB by atropine, utilizing a filtration method to divide edge QNB from free QNB. Radiation on the filters will be measured by scintillation numeration and, after rectification for numbering efficiency, will be converted into molar units from specific radiations.

Introduction:

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Receptors for acetylcholine are present in many tissues and can be characterised as falling into two chief types, muscarinic or nicotinic, on the footing of their ability to adhere muscarine or nicotine severally. Several substances are known that bind to the muscarinic acetylcholine receptor ( mAChR ) : some of these are agonists ( which bind and arouse a response ) and some are adversaries ( which bind but do non arouse a response ) . In general, adversaries are used to mensurate receptor adhering as they bind with a higher affinity ( lower KD ) than agonists bind.

In this experiment you will look into some of the belongingss of mAChR in rat encephalon membranes by agencies of 3H-quinuclidinyl benzilate ( 3H-QNB ) binding.

This experiment is based upon an article by Yamamura & A ; Snyder ( 1974 ) Proc Natl Acad Sci USA 71: 1725-1729 ( See class website. )

Requirements:

1. Rat encephalon membranes – shop on ice. ( See P for readying method ) .

2. Sodium K phosphate ( NaKP ) 50 millimeter pH 7.4

standard 3H-QNB/NaKP assay mix ( NaKP + 1.3nM 3H-QNB,

11.2 ten 102 Bq/pmol

high concentration 3H-QNB/NaKP assay mix ( NaKP + 6.5 nM 3H-QNB,

11.2 ten 102 Bq/pmol

atropine solution ( 10 I?M – MW 290 )

* QNB AND ATROPINE ARE TOXIC SO HANDLE WITH CARE *

3. Small glass trial tubing, micropipettes – 200 I?l ( YELLOW TIPS ) , 1000 I?l ( BLUE TIPS ) , 5000 I?l ( WHITE TIPS )

4. Multiplex filtration setup + GF/C glass fiber filters ( 2.5 centimeter diam ) + forceps

5. Scintillation mini-vials + ‘Ultima Gold ‘ scintillant

Methods:

All checks have a concluding volume of 2.0 milliliter, made up of 1.5 ml 3H-QNB check mix, 0.3 ml H2O or atropine. The check is started by adding 0.2 ml membranes. The extra atropine added to the controls displaces the particular and saturable ( i.e. receptor-bound ) QNB go forthing the non-specific, non-saturable QNB edge to the membranes.

The checks are left for the appropriate length of clip, stopped by adding 2.0 ml NaKP to increase the volume and filtering instantly through glass fiber filters. These are washed with NaKP and counted overnight in a scintillation counter.

Day 1

1. Make up two ‘bulk ‘ checks, one to mensurate entire QNB binding ( with H2O ) and one to mensurate non-specific binding ( with atropine ) . Set up two 50 milliliters conelike flasks therefore:

A

Bacillus

3H-QNB ( 1.3 nanometer )

30.0 milliliter

30.0 milliliter

H2O

6.0 milliliter

0.0 milliliter

atropine

0.0 milliliter

6.0 milliliter

( this is adequate for 20 checks – you will make 18 checks )

2. Put up a filter tower with six GF/C filters. When you are ready, rapidly add 4.0 milliliters swirled membranes to each flask and whirl to blend.

3. Now remove 2.0 ml aliquots to filters, three for each flask, doing certain 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 atropine

it will wholly get rid of all binding

Filter rapidly through fresh GF/C filters.

4. Wash each filter with 5 milliliters NaKP, take filters to mini-vials, add 5 milliliter scintillant, invert, leave at least 1 hour, invert once more and number the radiation in the scintillation counter.

5. Repeat steps 3 & A ; 4 at times =10, 20, 30, 45 and 60 mins.

6. Using the swabs provided, take six separate samples to look into for radioactive taint, for illustration by rubbing baseball mitts, bench or anything that might hold been in contact with 3H-QNB. Carefully note the beginning of each swab. Then put each swab into a separate phial incorporating 5 milliliter of scintillant, as earlier, record the intervention of each, and direct them for numbering. This is a standard safety process when covering with radioactive chemicals. The sums of tritium involved in this experiment are improbable to damage your wellness. Nevertheless this is a utile exercising to happen prove your technique before you make a error with 32P or 125I ( much more detrimental ) .

Day 2

Note that you need to take great attention to acquire the right volumes of each solution into the appropriate tubings. The more attention you take, the better will be your consequences

Determine IC50 for atropine ( i.e. that atropine concentration which displaces 50 % of QNB adhering ) .

Take 5 little glass trial tubings ( 1-5 ) and set 1200 I?l of distilled H2O in each.

Now add 300 I?l of 10 I?M atropine to Tube 1, mix good and reassign 300 I?l to Tube 2.

Mix good and reassign 300 I?l to Tube 3.

Repeat up to Tube 5.

Calculate the atropine concentration in each tubing.

Set up 7 triplicate glass tubings ( A1, A2, A3, B1… … G3 ) as follows:

Tubes

300ml of

1.3 nM QNB assay mix

A

10mM atropine

1500ml

Bacillus

Tube 1

1500ml

C

Tube 2

1500ml

Calciferol

Tube 3

1500ml

Tocopherol

Tube 4

1500ml

F

Tube 5

1500ml

Gram

distilled H2O

1500ml

Equally quickly as possible attention deficit disorder 200ml membranes to each tubing. Proceed as described in 2 ) … .4 ) above, utilizing the incubation clip you calculated from Day1 ‘s experiment ( it should be at least 45 proceedingss ) . It is best to get down the reactions in two batches, with 5 proceedingss between each batch to let you clip to filtrate the first batch before the 2nd batch is due.

Calculate the mean radiation edge to each triplicate set of filters and change over this value into suited units of QNB edge ( nanomoles or picomoles ) . Plot these values against log10 [ atropine ] . Estimate the IC50 from the center of the curve and compare your consequence with that obtained by Yamamura & A ; Snyder.

While you are waiting for the reactions to make equilibrium, carry out a Lowry check for protein ( see P ) so that you can cipher specific QNB binding in fmol QNB per milligram protein, and compare your value to that given in the Yamamura & A ; Snyder paper.

You will be told in the category what quantities of membrane readying to utilize in this check.

Day 3

Note that you need to take great attention to acquire the right volumes of each solution into the appropriate tubings. The more attention you take, the better will be your consequences

Determine KD for QNB. You will do lower concentrations of QNB by thining the standard QNB assay mix with NaKP ; higher concentrations can be made from the high concentration 3H-QNB mix but this is purely limited at 20 checks per group – do n’t blow it.

Label eight trial tubings 1-8.

Tube

1.3 nM QNB mix

6.5 nM QNB mix

NaKP

milliliter

milliliter

Milliliter

1

0

7.50

0.00

2

0

2.50

5.00

3

0

5.00

2.50

4

0

3.20

4.30

5

6.00

0.00

0.00

6

2.50

0.00

5.00

7

5.00

0.00

2.50

8

3.50

0.00

4.00

Label eight sets of triplicate tubings A1, A2, A3… .H3. Add the H2O or atropine last.

Tubes

1500 I?l from Tube #

300 I?l

A

1

Water

Bacillus

2

Water

C

3

Water

Calciferol

4

Water

Tocopherol

5

Water

F

6

Water

Gram

7

Water

Hydrogen

8

Water

Now label a separate set of eight tubings label A4, B4aˆ¦H4. Set these up as the old but add Atropine alternatively of H2O. Note that this set is non done in triplicate.

Add 200 I?l of membrane readying to each tubing. Incubate the tubings as described in 2 ) … 4 ) above, the incubation clip being that determined on Day 1. It is best to get down the reactions in two batches with 5 proceedingss between to let you clip to filtrate the first batch before the 2nd batch is due.

Calculate the mean radiation edge to each triplicate set of filters and change over it into sums of QNB ( nano- or picomoles ) . Pull a consecutive line through the atropine controls, and deduct the values for each existent or ‘estimated ‘ atropine control from the ‘water ‘ values and utilize these informations to cipher the edge and free QNB values.

While you are waiting for the reactions to make equilibrium, carry out a Lowry check for protein ( P ) so that you can cipher specific QNB binding in fmol QNB per milligram protein, and compare your value to that given in the Yamamura & A ; Snyder paper.

The information from this experiment may be analysed by Scatchard analysis. This will be discussed during the undermentioned session. Further information about this and other methods of analysis can be found at:

hypertext transfer protocol: //www.curvefit.com/introduction75.htm

Dispose of your radioactive equipment and toxic chemicals in the right topographic points.

Datas analysis

Questions to believe about:

How many dpm should be present in each check? ( Calculate this. )

What is the likely nature of the non-specific binding?

Remark on the rate of adhering for the particular and the non-specific binding.

What other methods are available for mensurating 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 suited?

Does the QNB concentration affect the IC50 of atropine?

LOWRY ASSAY FOR PROTEIN

Reagent 1:

0.5 ml Cu tartrate has been mixed with 50 milliliters alkalic carbonate on the twenty-four hours of usage.

Cu tartrate ( 0.1 g CuSO4.5H2O added to 0.2 g NaK tartrate in 20 ml H2O )

alkaline carbonate ( 2 g NaOH in 20 ml H2O and adding 10 g Na2CO3, made up to 100 milliliters with H2O )

Reagent 2:

Commercial Folin-Ciocalteau reagent 1:1 in H2O

Method:

In a series of trial tubings, add the volume of membrane announced at the start of the category and do this up to 1 milliliters with H2O.

Prepare tubes incorporating 0, 50, 100 150 and 200 I?g bovine serum albumen ( BSA ) made up to 1 milliliter H2O. The concentration of BSA you are supplied with is 1 mg.ml-1.

Add 1.5 ml Reagent 1.

Mix good and go forth to stand for 10 min at room temperature.

Add 0.3 ml Reagent 2, mix good and go forth for 30 min.

Read at 660 nanometers.

Plot the information from the standard BSA tubings and cipher the protein concentration in the membranes.

Preparation OF RAT BRAIN TISSUES

Rat encephalon membranes for QNB adhering experiment

Rat encephalons were homogenised in 10 volumes ice-cold 0.32 M sucrose/0.1 millimeter PMSF with a Teflon-glass Potter homogeniser. This was centrifuged at 12000g x 10 proceedingss and the pellet resuspended in original volume of sucrose and frozen in aliquots.

( PMSF = phenylmethylsulphonylfluoride – half life in H2O c. 3hr )