Computer
Introduction to Neurotransmitters
using AChE
16
co
py
Neurons, the cells of the brain, communicate with each other and the rest of the body by releasing
neurotransmitters. Neurotransmitters are small chemicals that bind to receptors on other neurons,
cells, or tissues of the body. When a neurotransmitter binds to a receptor, a cellular response is
produced in the target cell. If enough target cells are activated, a physiological response is
produced in the body. If the neurotransmitter produces an increase in a physiological response,
we refer to it as an excitatory neurotransmitter. If the neurotransmitter produces a decrease in a
physiological response, we refer to the neurotransmitter as inhibitory. The physiological effect
produced by a neurotransmitter is terminated, in large part, by the action of enzymes that break
down the neurotransmitter.
Ev
al
ua
tio
n
The neurotransmitter acetylcholine (ACh) is an excellent example of a neurotransmitter that can
be either excitatory or inhibitory. ACh is one of the primary neurotransmitters of the peripheral
nervous system. The skeletal muscles of your body and the cardiac muscles of your heart are all
controlled by neurons that release ACh. Your brain makes your skeletal muscles move by
activating neurons that release ACh on your muscle cells. In contrast, your heart is inhibited by
neurons that release ACh. If these neurons become more active, and release more ACh, your
heart slows down. If these neurons become less active, and release less ACh, your heart will
speed up. Skeletal and cardiac muscles cells also contain an enzyme called acetylcholinesterase
(AChE). This enzyme rapidly breaks down ACh into the compounds acetate and choline,
terminating the action of the neurotransmitter (see Figure 1).
Figure 1
Acetylcholine is also a very important neurotransmitter in the central nervous system. In
Alzheimer’s disease, neurons in the brain that release ACh die. The death of these neurons
decreases the level of ACh in the brain. This decrease in ACh is thought to cause some of the
symptoms of Alzheimer’s disease. The drug tacrine is used to treat Alzheimer’s disease. Tacrine
inhibits the activity of AChE (see Figure 1). This causes an increase in the level of ACh in the
brain, and alleviates some of the symptoms of Alzheimer’s disease. However, tacrine also
inhibits AChE found in skeletal and cardiac muscles, and can produce some unwanted side
effects.
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This exercise is designed to introduce you to the pharmacology of neurotransmitters. It is very
difficult to study neurotransmitters directly. In many cases, the activity of an enzyme that
produces or breaks down a neurotransmitter is used instead. A simple method for assaying the
activity of AChE is the Ellman method. The compounds acetylthiocholine iodide (ACTHi) and
dithiobisnitrobenzoate (DTNB) are added to a solution containing AChE. AChE breaks ACTHi
down into acetate and thiocholine. Thiocholine then reacts with DTNB to form a compound
called 5-thio-2-nitrobenzoate (TNB). TNB is a yellow-colored compound with a peak absorbance
at 412 nm, which can be monitored using a Spectrometer or Colorimeter.
Your instructor has homogenized heart tissue and filtered the solution through cheesecloth. A
centrifuge was then used to isolate different fractions from the original homogenate. The nuclear
fraction should contain cell nuclei and other remnants of cardiac cells. The supernatant should
contain components from the extracellular fluid, membrane and intracellular contents of the cells.
In the first part of this activity, you will use the Ellman method to determine which fraction has
the greatest amount of AChE activity.
In the second part, you will create a dose-response curve for the compound tacrine. A dose
response curve is a graph that shows how increasing concentrations of a compound change a
biochemical or physiological process. A dose-response curve for an inhibitor is shown in
Figure 2. The graph has been normalized to the activity of a control that does not contain the
inhibitor. As the concentration
of the inhibitor increases, the
activity decreases. The
concentration that inhibits 50%
of the activity of the control is
called the IC50. This is a
parameter that pharmacologists
use to classify different
compounds. Note that the shape
of the dose-response curve is
shaped like a backward letter S.
However, the center of the
dose-response curve, which
contains the IC50, is usually
linear. You will use the linear
portion of the dose-response
graph to estimate the IC50 for
your data.
Figure 2
OBJECTIVES
In this experiment, you will
• Observe the reaction rate of acetylcholinesterase (AChE) found in heart tissue.
• Compare the reaction rate of AChE from different fractions of heart tissue.
• Observe the effect that the compound tacrine has on the reaction rate of AChE.
• Generate a dose-response curve for the compound tacrine.
• Estimate the effective IC50 of tacrine on heart AChE.
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Advanced Biology with Vernier
Introduction to Neurotransmitters using AChE
MATERIALS
computer
0.1 M phosphate buffer, pH 7.9
Vernier computer interface*
2 mL 10 mM DNTB
Logger Pro
2 mL 100 mM ACTHi
Colorimeter or Spectrometer
2 mL 1 mM Tacrine
4 plastic cuvettes with caps
2 mL whole extract (on ice)
20–200 µL micropipette**
2 mL supernatant (on ice)
100–1000 µL micropipette**
2 mL nuclear fraction (on ice)
200 µL micropipette tips (1 box)
2 mL dH20 (on ice)
1000 µL micropipette tips (1 box)
eight 15 mL centrifuge tubes
*No interface is required if using a Spectrometer.
**Appropriate graduated transfer pipettes (1 mL and 5 mL) may be substituted.
PROCEDURE
Both Colorimeter and Spectrometer Users
1. Obtain and wear goggles and gloves.
2. Use a 15 mL centrifuge tube to obtain 10 mL of 0.1 M phosphate buffer, pH 7.9.
3. Prepare a blank by filling a cuvette with 2 mL of phosphate buffer. To correctly use cuvettes,
remember:
•
Wipe the outside of each cuvette with a lint-free tissue.
• Handle cuvettes only by the top edge of the ribbed sides.
• Dislodge any bubbles by gently tapping the cuvette on a hard surface.
• Always position the cuvette so the light passes through the clear sides.
4. Add the following to the blank: 100 µL from the nuclear fraction, 100 µL of DTNB solution
and 100 µL dH20.
Spectrometer Users Only (Colorimeter users proceed to the Colorimeter section)
5. Use a USB cable to connect the Spectrometer to your computer. Choose New from the File
menu.
6. Calibrate the Spectrometer.
a. Place the blank cuvette into the cuvette slot of the Spectrometer.
b. Choose Calibrate ►Spectrometer from the Experiment menu. The calibration dialog box
will display the message: “Waiting 90 seconds for lamp to warm up.” After 90 seconds,
the message will change to “Warmup complete.”
c. Click Finish Calibration and allow the calibration to finish. Click
.
7. Determine the optimum wavelength for examining the absorbance and set up the datacollection mode.
a. Empty the blank cuvette. Fill a new cuvette with 2 mL of phosphate buffer.
b. Add 100 µL of whole extract, 100 µL of DNTB solution, and 100 µL of acetylthiocholine
iodide solution (ACTHi) to the cuvette.
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c. Cap the cuvette and gently invert the cuvette three times. Let the cuvette sit for 5 minutes
and then place the cuvette into the Spectrometer.
d. Click
. A full spectrum graph of the solution will be displayed. Note that one area
of the graph contains a peak absorbance. Click
to complete the analysis.
e. Store your data by choosing Store Latest Run from the Experiment menu.
f. To set up the data collection mode and select a wavelength for analysis, click Configure
Spectrometer Data Collection, .
g. Select Abs vs. Time as the Collection Mode. The wavelength of the maximum absorbance
(λ max) will be selected. Verify that the maximum absorbance is close to 412 nm. Click
.
h. Choose Data Collection from the Experiment menu. Change the data-collection length to
5 minutes. Change the data-collection rate to 30 samples/minute. Click
.
i. Remove the cuvette from the Spectrometer and dispose of the solution as directed and
proceed to Step 8.
Colorimeter Users Only
5. Connect the Colorimeter to the computer interface. Prepare the computer for data collection
by opening the file “16a Neurotransmitters” from the Advanced Biology with Vernier folder
of Logger Pro.
6. Open the Colorimeter lid, insert the blank, and close the lid.
7. To calibrate the Colorimeter, press the < or > button on the Colorimeter to select the
wavelength of 430 nm (Blue). Press the CAL button until the red LED begins to flash and then
release the CAL button. When the LED stops flashing, the calibration is complete. Remove
the cuvette from the Colorimeter and proceed to Step 8.
Both Colorimeter and Spectrometer Users
Part I Comparison of heart acetylcholinesterase activity from different fractions
8. Obtain 4 cuvettes and label the caps.
Cuvette 1 = N
Cuvette 2 = S
Cuvette 3 = W
Cuvette 4 = C
9. Fill Cuvettes 1–4 with 2 mL of 0.1M phosphate buffer.
10. Do this quickly! Add 100 µL of whole extract, 100 µL of DNTB solution, and 100 µL of
acetylthiocholine iodide solution to Cuvette 1.
11. Cap the cuvette and gently invert the cuvette three times. Place it in the device (close the lid
if using a Colorimeter). Click
. Absorbance data will be collected for 5 minutes.
Discard the cuvette contents as directed by your instructor at the end of the run.
12. Store your data by choosing Store Latest Run from the Experiment menu.
13. Do this quickly! Add 100 µL of supernatant, 100 µL of DNTB solution, and 100 µL of
ACTHi to Cuvette 2.
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Advanced Biology with Vernier
Introduction to Neurotransmitters using AChE
14. Repeat Steps 11–12.
15. Do this quickly! Add 100 µL from the nuclear fraction, 100 µL of DNTB solution, and
100 µL of ACTHi to Cuvette 3.
16. Repeat Steps 11–12.
17. Do this quickly! Add 100 µL from the nuclear fraction, 100 µL of DNTB solution, and
100 µL of dH20 to Cuvette 4.
18. Repeat Steps 11–12.
19. On the graph, select the most linear region of all data, typically between minutes 1 and 4.
20. Click Linear Fit, . Select all runs and click
shown for each run selected.
. A best-fit linear regression line will be
21. Record the value of the rate (slope), m, for each run in Table 1.
22. (Optional) Print or choose Save As from the File menu to save your data.
Part II Increasing enzyme concentration
23. Choose Clear All Data from the Data menu.
24. Using 1 mM tacrine as a stock, create 10 mL of the following solutions: 1×10-5, 1×10-7,
1×10-9, 1×10-11, and 1×10-13 M tacrine in 0.1 M phosphate buffer. The best way to accomplish
this is to perform a set of serial dilutions. Ask your instructor if you have questions about
how to prepare these solutions.
25. Obtain 6 cuvetttes with caps.
a.
b.
c.
d.
e.
f.
Label the cap of Cuvette 1 with a C and add 2 mL of 0.1M phosphate buffer.
Label the cap of Cuvette 2 with a –13 and add 2 ml of 1×10-13 M tacrine.
Label the cap of Cuvette 3 with a –11 and add 2 ml of 1×10-11 M tacrine.
Label the cap of Cuvette 4 with a –9 and add 2 ml of 1×10-9 M tacrine.
Label the cap of Cuvette 5 with a –7 and add 2 ml of 1×10-7 tacrine.
Label the cap of Cuvette 6 with a –5 and add 2 ml of 1×10-5 tacrine.
26. Select Cuvette 1.
27. Do this quickly! Add 100 µL of whole filtrate, 100 µL of DNTB solution and 100 µL of
ACTHi.
28. Cap the cuvette and gently invert it three times. Place it in the device (close the lid if using a
Colorimeter). Click
. Absorbance data will be collected for 5 minutes. Discard the
cuvette contents as directed by your instructor at the end of the run.
29. Store your data by choosing Store Latest Run from the Experiment menu.
30. Repeat Steps 27–29 for Cuvettes 2–6.
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31. Select all of the data on the graph. Click the Linear Fit button, . Select the correct runs and
click
. A best-fit linear regression line will be shown for each run selected.
32. Record the value of the rate, m, for each of the 6 runs in Table 2.
DATA
Part I Comparison of heart acetylcholinesterase activity from different fractions
Table 1
Source
Rate
(Δ abs/min)
Whole filtrate
Percent difference
100%
Supernatant
Nuclear fraction
Control
Part II Effect of tacrine on heart acetylcholinesterase activity
Table 2
Tacrine concentration
(M)
Rate
(Δ abs/min)
Normalized activity
(%)
Control (no tacrine)
-13
1x10
-11
1x10
-9
1x10
-7
1x10
-5
1x10
Est. IC50
_____________________
PROCESSING THE DATA
Part I Comparison of heart acetylcholineseterase activity from different cellular fractions
1. Calculate the percent difference in activity for each sample. The rate for the whole filtrate is
considered 100%. Divide the rate for each fraction by the rate of the whole filtrate. Multiply
this number by 100 to get the percent difference from the filtrate. Record this value in the
space provide in Table 1.
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Advanced Biology with Vernier
Introduction to Neurotransmitters using AChE
2. Create a bar graph of your raw data using Logger Pro.
a. Open the file “16b Neurotransmitters” from the Advanced Biology with Vernier folder of
Logger Pro.
b. Enter the slopes you observed for each fraction in rows 1–4 of the Reaction Rate column.
Part II Effect of tacrine on heart acetylcholinesterase activity
3. Calculate the normalized activity rate for each concentration of tacrine. The rate for the
control in Table 2 is considered 100%. Take the rate observed for each tacrine concentration
and divide it by rate observed for the control. Multiply this number by 100 to get the
normalized activity rate for each concentration. Record the normalized response for each
tacrine concentration in the space provide in Table 2.
4. Create a dose-response graph of your data using Logger Pro.
a. Click Next Page, , to go to the next page of the file.
b. Enter the percent activity that you calculated for each tacrine concentration in rows 1–6 of
the Normalized Activity column. These values are located in Table 2.
c. Drag your cursor across the central, linear portion of the data. The curve fit from this
portion will be used to estimate IC50 for your data.
d. Click Curve Fit, .
e. Choose Base-10 Logarithm (y = A*log(B*x)) as the General Equation. Click
.
f. A best-fit curve for the selected data points will be displayed on the graph. Click
.
5. Choose Interpolate from the Analyze menu and find the point on your graph that corresponds
to, or is closest to, a normalized activity rate of 50%. Record the corresponding concentration
using scientific notation in the space provided in Table 2. This is your estimate of the IC50 of
tacrine for your heart tissue extract.
6. (Optional) Save your file. Insert a Text Annotation that corresponds to your estimate of the
IC50 and print copies of your graphs.
QUESTIONS
Part I Comparison of heart acetylcholinesterase activity from different fractions
1. Was the change in absorbance faster for the nuclear fraction or the supernatant? Which
fraction contains the greatest amount of acetylcholinesterase based on your data?
2. How many times faster was the change in absorbance for the whole filtrate when compared to
the supernatant and the nuclear fraction? If you add the rates for the supernatant and the
nuclear fraction together do they equal the rate observed for the whole filtrate.
Part II. Effect of tacrine on heart acetylcholinesterase activity
3. How does increasing the concentration of tacrine affect the change in absorbance?
4. Were you able to estimate the IC50 of tacrine for your sample? How does the IC50 of your
sample compare to data for other students.
5. If tacrine was given to a person, can you explain what would happen to levels of
acetylcholine in the heart, skeletal muscles and brain? Can you explain how this would
happen at the cellular or molecular level?
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EXTENSIONS
1. Calculate the reaction rate of TNB production for each run using the formula below. This
value corresponds to the actual enzymatic rate of the acetylcholinesterase in the heart extract.
Reaction rate = (Δ abs/min)/(1415 M-1 cm-1) x 1 cm.
2. Calculate the specific acetylcholinesterase activity of the heart extract for each part of this
exercise. Specific enzyme activity can be defined as the reaction rate of the extract for a
known concentration of substrate divided by the amount of protein found in 1 mL of the
extract. The whole filtrate was prepared at a concentration of 100 mg tissue/mL buffer. Use
the “Got Protein? Kit” from Bio-Rad Laboratories Inc. (Catalog # 166-2900EDU) to
determine the actual protein concentration of the whole filtrate and each fraction.
3. Calculate the IC50 of tacrine from your data and compare this value to your estimate of the
IC50. Take the log of the tacrine concentrations from Part II (located in Table 2) and record
these values. Follow the instructions below to create a new dose-response curve using
Logger Pro.
a. Open a new, blank file in Logger Pro or, if you have a file that was created while doing
Experiment 16, choose Add Page from the Page menu and choose New Data Set and
.
Graph as the Starting Contents. Click
b. In the data table, double-click the heading, Data Set, and rename the data set Dose
Response for Tacrine.
c. Double-click the heading of the X column.
d. Enter Log Concentration as the Name and Log Conc as the Short Name. Leave Units
blank. Click
.
e. In the Log Concentration column, enter the log of the concentration of tacrine values that
you calculated above.
f. Double-click the heading of the Y column.
g. Enter Normalized Activity as the Name, % Act as the Short Name, and % as the Units.
Click
.
h. In rows 1–5 of the Normalized Activity column, enter the percent activity that you
calculated for each tacrine concentration. These values are located in Table 2.
i. Autoscale the graph.
j. Click and drag on the graph to select the portion of your data that is now linear.
k. Click Linear Regression, . Using the linear regression formula, y = mx + b, solve for x
where y = 50%. Record the value for x.
l. Calculate the anitlog, 10x, to determine the calculated value for IC50.
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4. Estimate the IC50 of tacrine from your data using the Four Parameter Log Model and compare
this estimate to your original estimate of the IC50. Take the log of the tacrine concentrations
from Part II and record these values. Follow the instructions below to create a new doseresponse curve using Logger Pro.
a. Open a new, blank file in Logger Pro or, if you have a file that was created previously for
this activity, choose Add Page from the Page menu and choose New Data Set and Graph
as the Starting Contents. Click
.
b. In the data table, double-click the heading, Data Set, and rename the data set Dose
Response for Tacrine.
c. Double-click the heading of the X column.
d. Enter Log Concentration as the Name and Log Conc as the Short Name. Leave Units
blank. Click
.
e. Enter the log of the concentration of tacrine used for each run in rows 1-5 of the Log
Concentration column. These values are located in Table 2.
f. Double-click the heading of the Y column.
g. Enter Normalized Activity as the Name, % Act as the Short Name, and % as the Units.
.
Click
h. Enter the percent activity that you calculated for each tacrine concentration in rows 1–5 of
the Normalized Activity column. These values are located in Table 2.
i. Autoscale the graph.
j. Click Curve Fit, , and then click the Define Function button.
k. Enter a+(b–a)/(1+10^((x–c) x d)) as f(x) and Four Parameter Log Model as the
Description. Click
.
l. Click
. A best-fit curve will be displayed on the graph. The curve should match up
well with the points. If the curve has a good fit with the data points, then click
.
m. Choose Interpolate from the Analyze menu and find the point on your graph that
corresponds to, or is closest to, a normalized activity rate of 50%. This is your estimate of
the IC50 of tacrine for your heart tissue extract.
5. Repeat your dose-response curve for tacrine using a larger number of concentrations. Start at
1×10-15 M tacrine and proceed in 10X steps to 1×10-3 M tacrine. Repeat the experiment
3-5 times. Make sure you also run the appropriate number of controls. Calculate the average
response for each concentration. Convert each average response to an actual reaction rate. See
Extension 1. Normalize your data, plot a new dose-response curve and estimate or calculate
the IC50 of tacrine using the instructions in Extension 3.
6. Design an experiment that will determine the Km of acetylcholinesterase from your heart
tissue homogenate. Ask your instructor how to proceed.
Advanced Biology with Vernier
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z
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Essential instructor background information
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The complete Advanced Biology with Vernier lab manual includes 27 labs and essential
teacher information. The full lab book is available for purchase at:
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