MONOAMINE OXIDASE FROM HUMAN BRAIN: ISOLATION, CHARACTERIZATION AND SUICIDE INHIBITION.
Item
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Title
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MONOAMINE OXIDASE FROM HUMAN BRAIN: ISOLATION, CHARACTERIZATION AND SUICIDE INHIBITION.
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Identifier
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AAI8801765
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identifier
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8801765
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Creator
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STRAHER, MICHAEL PAUL.
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Contributor
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Morton D. Glantz
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Date
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1987
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry
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Abstract
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Monoamine Oxidase (E.C. 1.4.3.4) was purified to apparent homogeneity from human brain mitochondria by a procedure which included Triton X-100 extraction, ammonium sulfate fractionation, Sepharose DEAE-CL-6B chromatography and Bio-Gel HTP chromatography.;The final purified enzyme has a fold purification of 21.4. The enzyme traveled as a single band in 4% polyacrylamide electrophoresis gels, and showed minimum native molecular weights of 109,600 and 102,000 in Sepharose CL-6B and Waters I-300 HPLC permeation chromatography systems, respectively. Pargyline titration indicated that the enzyme as isolated was exclusively type B.;Amino acid analysis of the enzyme revealed alanine and glycine to be the most abundant residues. Particle Induced X-Ray Emission (PIXE) analysis of the enzyme indicated the relative absence of iron and copper, supporting the concept that MAO is not a metal dependent enzyme.;Michaelis constants for benzylamine and kynuramine were found to be 145 {dollar}\mu{dollar}M and 63 {dollar}\mu{dollar}M, respectively, and the respective pH optima for the substrates were 9.0 and 8.8.;Three new N-cyclopropyl inhibitors of MAO were synthesized. The compounds, p-hydroxy N-cyclopropylbenzylamine, p-methoxy N-cyclopropylbenzylamine and p-methoxy N-cyclopropyl {dollar}\beta{dollar}-phenethylamine, were demonstrated to be suicide inhibitors of MAO from human and pig brain. Ki values for the three inhibitors were determined for MAO from human brain (p-hydroxy N-cyclopropylbenzylamine, 4.5mM) and pig brain (p-methoxy N-cyclopropylbenzylamine and p-methoxy N-cyclopropyl {dollar}\beta{dollar}-phenethylamine, 516 {dollar}\mu{dollar}M and 833 {dollar}\mu{dollar}M, respectively). The inhibition caused by all three compounds was found to be completely reversible upon dialysis against 1.0 mM benzylamine.;The use of p-hydroxy N-cyclopropylbenzylamine as an affinity ligand to aid in the isolation of MAO was explored. The ligand was bound to epoxy-activated Sepharose CL-6B, and the resulting gel was observed to bind partially purified pig brain MAO. Upon elution with 1.0 mM benzylamine, a small amount of enzyme was recovered with an eight fold increase in specific activity.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biochemistry
