The effect of lipid environment on the mitochondrial monoamine oxidase of bovine liver.
Item
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Title
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The effect of lipid environment on the mitochondrial monoamine oxidase of bovine liver.
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Identifier
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AAI9618055
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identifier
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9618055
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Creator
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Deery, Ann Marie.
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Contributor
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Adviser: Lesley Davenport
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Date
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1996
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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 | Biology, Cell | Biology, Animal Physiology
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Abstract
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The aims of this investigation were the isolation of bovine mitochondrial MAO-B, its reconstitution into vesicles of defined lipid composition, and examination of the influence of different lipid environments on its enzymatic activity.;Of several isolations, one preparation was purified to homogeneity, two others purified to {dollar}\geq{dollar}80% with high specific activity, while other preparations were less pure. Enzymatic activity was assayed spectrophotometrically with kynuramine. Triton X-100 was included in the assay of the membrane-free ("solubilized") enzyme to prevent aggregation. MAO-B was reconstituted by octylglucoside dialysis. On average, 96% of the protein recovered on glycerol gradients was vesicle-incorporated. The influence of phospholipid headgroup and fatty acyl chain composition were investigated. MAO-B was reconstituted with a range of concentrations of dioleoylphosphatidylethanolamine (DOPE) in dioleoylphosphatidylcholine (DOPC). Activity and stability increased with increasing DOPE content, reaching a maximum at about 40-50 mole%. Proteoliposomes (PRLs) composed of 100% DOPC and DOPC/DOPE (70/30 mole%) were selected for further study. The effects of membrane surface charge due to the anionic lipids, dioleoylphosphatidylserine (DOPS) and dioleoylphosphatidic acid (DOPA), were examined. PRLs were prepared containing the anionic species either as the sole lipid or as 10 mole% with DOPC or the DOPC/DOPE mixture. Whereas 10 mole% DOPS did not directly affect MAO-B activity, its stability was decreased. In contrast, this concentration of DOPA significantly enhanced the enzymatic activity. The effects of 10 mole% anionic lipid were reduced when 30 mole% DOPE was also present. The degree of unsaturation of the 18 carbon fatty acyl chains of PC did not alter the activity. The kinetics of kynuramine oxidation were compared for membrane-free enzyme and the DOPC/DOPE (70/30 mole%) reconstituted system. The K{dollar}\rm\sb{lcub}M{rcub}{dollar} was unaltered; however, the V{dollar}\rm\sb{lcub}max{rcub}{dollar} for the PRLs was 60% of the value for solubilized enzyme.;In summary, there was no dramatic increase in MAO-B activity upon reconstitution. It may be that this lack of sensitivity to its lipid environment is important for MAO-B function. The membrane may serve to regulate activity, preventing oxidative stress from a build-up of toxic by-products.
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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.
