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Characterization of Inhibition of Monoamine Oxidase Activity

Characterization of Inhibition of Monoamine Oxidase Activity. Mark Delboy August 6, 2003. Progress Report. Goal of experiment- Overview: Main goal of present study:

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Characterization of Inhibition of Monoamine Oxidase Activity

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  1. Characterization of Inhibition of Monoamine Oxidase Activity Mark Delboy August 6, 2003

  2. Progress Report Goal of experiment- Overview: • Main goal of present study: • To establish methods for the measurement of simultaneous MAO and CYP3A4 enzyme activities by studying the metabolic kinetics. • Reactions schemes that were planned: 1.) kynuramine and MAO A. 2.) 7-benzyoxyquinolone and CYP3A4. 3.) kynuramine , MAO A and CYP3A4. 4.) 7-benzyoxyquinolone, MAO A and CYP3A4 kynuramine, MAO A, 7-benzyoxyquinolone and CYP3A4. • What was achieved this summer: 1.) kynuramine and MAO A.

  3. Progress Report Importance of MAO- Overview: • Monoamine oxidase (MAO) is a type of flavoenzyme involved in redox reactions in biological systems. • Involving the conversion of an amine substrate, such as neurotoxin, neurotransmitter, or exogenous material, to an aldehyde. • MAO has been found to be located in the mitochondrial outer membrane of platelets, in the liver and in the brain.

  4. Progress Report Importance of MAO- Overview: • Due to the location of MAO activity, some of the biochemical activity of the enzyme can lead to free radicals that can lead to neurodegenerative diseases. Enzyme Kinetics- Overview: • Ideally, the Michaelis-Menten equation is used for enzymes to describe the relationship between the substrate concentration and the metabolism rate and is often used for the MAO enzymes,

  5. Progress Report Fluorescence- Overview: • 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.

  6. Progress Report Results- Overview. • The method for measuring MAO-A activity was determined after numerous trials and two incubations. • The metabolic kinetics for the MAO-A and kynuramine system was then analyzed using Matlab to attain Km and Vmax. Results- Method of Measurement- Overview: • The wavelength of fluorescence emission was determined. • Appropriate concentration ranges were determined. • Calibration concentrations and curves were determined.

  7. Progress Report Results- Determination of emission wavelength of fluorescence emission: • With the emission obtained at an excitation of 310: • intensities of the signals were weaker. • various concentrations were condensed. • With the emission obtained at an excitation of 320: • intensities of the signals were stronger. • various concentrations were clearer.

  8. Progress Report Results:Determination of appropriate concentration ranges.

  9. Progress Report Results - Determination of calibration curves: • A calibration curve was made by plotting fluorescence intensity against the HQ concentration at 350 nm. • The following equation applies to the calibration curve : • m: slope of the line. b: is the y intercept. x: concentrations of the substrate. y: fluorescent signal values.

  10. Progress Report Results - Analysis using Matlab :

  11. Progress Report Results - Analysis using Matlab : Michaelis-Menten plots

  12. The Academic Year Goals for the experiment- Overview: • Problem: • A colleague of Dr. Castagnoli, Professor Edmondson found that the MAO-B expressed enzymes activity, from Gentest, had not been inhibited by chlorostyrylxanthinyl analog (CSC). • Castagnoli’s studies with the MAO-B liver mitchondrial enzyme preparation, however, indicate the inhibitor having a significant inhibition constant. • Conflicting results observed from expressed enzymes prepared from cDNA recombinant expression in baculovirus systems and enzymes prepared from mitochondrial membranes from the liver.

  13. The Academic Year Goals for the experiment- Overview: • Goal: • Determining if differences exist between the activities and inhibition of human expressed MAO-B and human liver mitochondrial MAO-B. • Relationship to current study: • Assumption of current study- mixtures of expressed enzymes suitably represent behavior of enzymes in vivo. • A difference in the enzymes would affect the validity of our current tests.

  14. The Academic Year Plans for the experiment- • Overview: • In order to resolve such problems, the values for Ki constants of a variety of inhibitorsshould be determined for both enzymes. • By measuring the extent that metabolite production is slowed down, inhibition patterns can be used to characterize the enzymes from the two sources. • Actual Procedure: • MAO-B SUPERSOMES, MAO insect controls, Human Liver Microsomes will be purchased from Gentest.

  15. The Academic Year Plans for the experiment- • Actual Procedure: • Benzylamine (Sigma) will be used as the substrate to observe the inhibition of MAO-B. • MPTP, Deprenyl, Amitriptyline, Fluoxetine, Paragyline, all known to be inhibitors with expressed MAO-B enzymes, will be tested. • The inhibition will be measured with the fluorescence spectrometer using the MAO-A method and detecting the product benzaldehyde. (except with an excitation wavelength of 310 nm with a emission wavelength range of 330 to 450 nm)

  16. The Academic Year Plans for the experiment- • Actual Procedure: • The various inhibitor concentrations will be run on both types of enzymes. • Matlab will then be used to analyze the data and obtain the values for the Michaelis constants and compare the two enzymes for differences.

  17. Thank you very much. Questiones?

  18. Acknowledgements Sarah Rutan and Ray Sanchez for the constant help, guidance, and persistence. Many thanks. Ernst and Sarah Graham for their help in lab when I was confused. Thanks my chums. Jeff Elhai for his comments, work put into the program and wisdom. Thanks many times over. The rest of you--thanks. For doing what you do best--being there.

  19. References 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/.

  20. References 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/ 12. An Introduction to Fluorescence Measurements. Turnerdesigns,Inc. 2002. http://www.turnerdesigns.com/ END

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