Introduction
Metabolism is the transformation of compounds produced within (endogenous)
and outside (exogenous or xenobiotic) of an
organism and the consequences that occur as these compounds are transported
and eliminated by biological systems (ISSX 1).
Both Monoamine Oxidase (MAO) is an enzyme that is involved in complex systems
of metabolism that can be studied by its
effects on reaction rates of substrate to product conversion in order to
find more information about how these systems work.
Monoamine oxidase (MAO) is a type of flavoenzyme involved in redox reactions
in biological systems (Castagnoli 2001;
Kawai 1996). A flavin (see Figure 1) is located near the active site and
aids the enzyme in catalytic activities (Kawai 1). The
redox reactions catalyzed by flavoenzymes involve the conversion of an amine
substrate to an aldehyde (Castagnoli 2001).
(Kawai 1).
Amine-containing compounds, including many neurotoxins, neurotransmitters,
and exogenous materials, such as pharmaceuticals,
mutagens, and pollutants can undergo oxidative deamination reactions catalyzed
by MAO (Castagnoli 2001, Lin 2001). MAO
has been found to be located in the mitochondrial outer membrane in either
A or B form; these forms are distinguished by
differences in their selectivity (Castagnoli 2001).The reactions with the
enzyme can be thought to follow the following
reaction scheme,
For the reaction scheme above, E is the participating enzyme, S is the substrate
being used, ES the enzyme-substrate complex
formed, and P is the final product (Clarke 1998). To describe the relationship
between the substrate concentration and the
metabolism rate, the Michaelis-Menten equation is often used for the MAO
enzymes,
In this formula, [S] is the substrate concentrations. The rate of the reaction
is ν while Vmax is a constant that is the maximum value
allowed for the rate. Km is another constant that describes the concentration
of the substrate at which v is 50% of Vmax
(Houston & Kenworthy 1999). When the metabolite kinetics follow the
Michaelis-Menten model, then plotting ν vs. [S] gives a
hyperbolic graph. In our experiments we will be assuming that the reactions
will follow the Michaelis-Menten equation (Houston
& Kenworthy 1999).
A fluorescence spectrometer (Varian Eclipse) will also
be used to determine the amount of product made over a certain time
interval. During fluorescence experiment, molecules absorb radiation
and are excited from the ground state to a higher energy
state. The molecules soon return to ground state but simultaneously
emit light with an energy less than or equal to the energy
absorbed. A portion of radiation that exits the cell containing the compound
is measured by a detector (Braun 1987). After
obtaining the data, a plot of the fluorescence intensity can be prepared
and the concentration of product can be obtained
(Braun 1987).
The MAO activity, which involves speeding up redox reactions,
is of particular interest due to MAO’s involvement in
neurodegenerative diseases. In studying the rate constants, we hope to take
progressive steps at towards preventing such
diseases as Parkinson’s.
Progress Report
A method for measuring MAO-A activity using fluorescence
spectroscopy was developed, based on the chromatographic
method described on the Gentest website (Gentest 1987).
A buffer was prepared from pure water with sodium phosphate
to obtain a solvent with a pH of 7.4. A stock solution for
KY was made using the buffer to obtain a solution of 250 µM. From
the stock solution, different concentrations of KY were
prepared and were added to individual spectrofluorimetric cuvettes. The
amounts of buffer and KY used in each trial are shown
in Tables 1 and 2,
The enzyme was then diluted to a concentration of 0.5
mg/mL and then 40 µL of the latter dilution was added to each incubation.
The final volume of solution to be tested in the cuvette was 3 mL. The cuvettes
were placed in the spectrofluorimeter for 20
minutes. A spectral measurement was made after a 2 minute delay time and
subsequently at1 minute intervals, during which time
a stir bar was used to insure a complete mixsed solution and the temperature
was held at 37oC. Fluorescence emission was
measured using an excitation wavelength of 320 nm with a emission wavelength
range of 330 to 550 nm. An incubation with 80
µM of KY and MAO insect control enzyme was also carried out. Then
solutions ranging from 0.5 mM to 10 mM of
4-hydroxyquinoline in phosphate buffer were used to obtain data to be used
for a calibration curve. A calibration curve was
made by plotting fluorescence intensity against the HQ concentration at
350 nm, the calibration curve equation needed for later
analysis was obtained.
The metabolic kinetic data for the MAO-A and kynuramine
system were then analyzed using Matlab. From the calibration curves,
an equation was found giving values for the slope and intercept. The equation
of the line is of the form , where m is the slope of
the line and b is the y intercept. The x is actually the concentrations
of the substrate, while the y is the fluorescent signal values
obtained during the incubation. The signal values obtained from the incubations
in the form of spectra for the various concentration
trials were taken from the 20 minutes time points for each trial. By rearranging
the equation, , the concentrations of the substrates
could be determined. Once these concentrations were found, a plot
of concentration against time was be made and the curve
that was observed was linear. The slopes of these curves were obtained,
giving the initial rates in units of uM/min. A plot of these
rates against [S] was made to show typical Michaelis-Menten behavior. Then
the nonlinear least-squares curve fitting, routine
from the Matlab optimization toolbox (under lsqcruvefit) was used to find
the function that best fit the data to the Michaelis-Menten
equation. Values for Km and Vmax were obtained from the fits for both incubations.
The hyperbolic graph and the graph from
the fit sre shown in Figures 3 and 4,
For the first incubation, the Michaelis constants were
found to be, Km= 18.9 µM and Vmax= 0.24 µM/min while the
second incubation had slightly higher constants, Km= 20.5 µM
and Vmax= 0.29 µM/min. The Matlab graphs show a high
reproducibility between the incubations, thus the resulting constants,
Km and Vmax, were similar values. The values obtained
in the incubations were lower than the literature values, Km= 40 µM
and Vmax= 67 nmol/mg/min (Gentest 1987). The reasons
for this discrepancy will be investigated in further studies With
a high reproducibility obtained, we are fairly content with the
validity of our method of analysis for the MAO-A and kynuramine system.
Goals for Academic Year
Professor Edmondson has obtained an X-ray structure for
MAO-B. He has found that the expressed enzymes activity,
enzymes produced by Gentest from cDNA using a baculovirus expression system,
had not been inhibited by chlorostyrylxanthinyl
analog (CSC). Castognoli’s studies with the liver mitchondrial
enzyme preparations, however, indicate the inhibator having a rate
constant (Ki) of about 100 nM (Nandigama 2002 and Petzer 2003). Determining
if difference exists between the activities of
human expressed MAO-B and human liver mitochondrial MAO-B is the particular
focus for studies during the upcoming
academic year. One reason that such a discrepancy needs to be investigated
is that the kinetic differences could relate to
structural features of the liver enzyme from the mitochondrial membrane
that are different from the Gentest solubilized enzyme.
This difference in the enzymes would affect the validity of our current
tests, since we are using Gentest enzymes and are assuming
that they function in similar ways to those within the body (Castagnoli
2003).
Plan for Academic Year Experiments
Neuron firing rates throughout the human body can be
kept at homeostatic levels by MAO creating bioactive amines in the
liver and metabolizing in the neurons of the brain (Richardson 1993). Due
to the location of MAO activity, some of the
biochemical activity of the enzyme creates can lead to free radicals that
can react and lead to neurodegenerative diseases
(Richardson 1993). As mentioned previously, MAO is currently being studied
at Prof. Castagnoli’s lab, with a focus on
preventing the formation of free radicals that may cause Parkinson’s Disease.
The inhibitor, 1-methyl-4-phenyl-1,2,3,6
tetrahydropyridine (MPTP), is of current interest. Prof. Castagnoli and
his colleague, Professor Edmondson, noticed a conflict
between the enzyme characteristics of the enzymes from the two sources.
This observation leads to the question of whether
Castagnoli’s current data is valid, based on the fact he is studying the
effect of inhibitors on the Gentest enzymes. In order to
resolve such problems, the values for Ki should be determined for both the
Gentest expressed enzyme and for the mitochondrial
liver enzyme. By measuring the extent that a metabolite is slowed down in
production, various known inhibitors can be tested
and the enzymes can be compared.
MAO-B Supersomestm, Human Liver Microsomes and MAO-B
insect controls (expressed enzymes) will be
obtained from Gentest, while other mitochondrial liver enzymes will be obtained
from colleagues of Dr. Castagnoli.
Benzylamine will be used as the substrate (Sigma) to observe the inhibition
of MAO-B. (Gentest 1987). The latter
compound is considered to be a selective substrate for MAO-B and this is
required when the mitochondrial liver enzymes
are studied, since there might also be MAO-A in the microsomes would obscure
the results (Gentest 1987).
The compounds to be used to test inhabitation, which
will more than likely come from colleagues but could also be
order from Sigma. The candidates for such inhibition testing include
MPTP, Deprenyl, Amitriptyline, Fluoxetine, and
Paragyline, all known to be inhibitors of Gentest MAO-B enzymes (Gentest
1987). The enzymes inhibition will be measured
with the fluorescence spectrometer, using nearly the same method as
was developed for the analysis of the MAO-A and
kynuramine system. A 7.4 phosphate buffer will be used to make
a 250 µM benzylamine stock solution, which will allow
different concentrations of benzylamine to be prepared. Each type
of enzyme will be used seperately to test various
concentrations of inhibitors. So diluted enzyme will then be added
accordingly to the other compounds to make 3 mL
cuvette solutions and measured over 20 minutes at 37oC with mixing
using a stir bar. Fluorescence emission from the
product benzaldehyde will be measured at an excitation wavelength
of 310 nm with a emission wavelength range of 330
to 450 nm. Incubations will also be carried out using the MAO insect
control enzymes will then be tested under the same
conditions. Then benzaldehyde solutions in phosphate buffer will be
used to obtain data to be used for a calibration curve.
Matlab will be used to analyze the data and obtain the values for
the Michaelis constants.
By the end of the academic year, data should be obtained to support
or refute the use of Gentest enzymes in experiments
used to simulate human metabolism.
Budget
2 Disposable fluorescence cuvettes- Methacrylate-
$ 208.60
MPTP - 20 mg.-
$ 90.40
Benzylamine- 100 mL-
$ 39.20
MAO-B SUPERSOMEStm -2 vials-
$ 500.00
Human Liver Microsomes- 1 vial-
$ 250.00
MAO Insect Control-1 vials-
$ 250.00
Estimated Total
$1338.20
References
Most certainly Dr. Sarah Rutan and Ray Sanchez. And also my thanks.
1. Castagnoli, N., Dalvie, D., Kalgutkar, A., & Taylor, T. Interactions
of Nitrogen Containing Xenobiotics with Monoamine
Oxidase (MAO) Isozymes A and B: SAR Studies on MAO Substrates and Inhibitors.
Chemical Research in Toxicology,
14 (9), pp. 1139 -1162, 2001, http://pubs.acs.org/.
2. Clarke, S. In Vitro Assessment of Human Cytochrome P450. Xenobiotica,
28,(12), 1998,
http://taylorandfrancis.metapress.com/.
3. Parikh, S., Hanscom, S., Gagne, P., Crespi, C., & Patten, C. A Fluroescent-Based,
High-Throughput Assay for Inhibitors
of Human Monoamine Oxidase A and B. Gentest. http://www.gentest.com/.
4. JS, Richard. On the Functions of Monoamine Oxidase, the Emotions, and
Adaptation to Stress. Int J Neurosci. Vol 70,
Issue1-2, May 1993, pp. 75-84. http://www.biopsychiatry.com/mao.htm/.
5. Kawai, Y., Kunitomo, J., & Ohno, A. Atropisomeric Flavoenzyme Models
with a Modified Pyrimidine. Kyoto: Institute
for Chemical Research – ICR Annual Report. 3, 1996, http://www.kuicr.kyoto-u.ac.jp/.
6. ISSX Constitution. International Society for the Study of Xenobiotics.
http://www.issx.org/.
7. Human Monoamine Oxidase A (MAO-A). Gentest. http://www.gentest.com/.
8. Braun, R. Introduction to Instrumental Analysis. McGraw-hill, pp.316-346;
821-869, 1987.
9. Jacobus P. Petzer, Salome Steyn, Kay P. Castagnoli, Jiang-Fan Chen,Michael
A. Schwarzschild, Cornelis J. Van der
Schyf and Neal Castagnoli. Inhibition of monoamine oxidase B by selective
adenosine A2A receptor antagonists. Bioorganic
& Medicinal Chemistry. Volume 11, Issue 7 , April 2003, pp. 1299-1310.http://www.sciencedirect.com/.
10. Ravi K. Nandigama, Paige Newton-Vinson and Dale E. Edmondson. Phentermine
inhibition of recombinant human liver monoamine oxidases A and B. Biochemical
Pharmacology. Volume 63, Issue 5 , 1
March 2002, Pages 865-869. http://www.sciencedirect.com/.
11. Abstract_4. Free presentation backgrounds. I to Eye. http://www.itoeyeonline.co.uk/
