Immunoreactivity of Lecithin: Cholesterol Acyltransferase (LCAT); A Tool for Measurement of Levels and Characterization

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dc.contributor.advisorLacko, Andras G.
dc.contributor.committeeMemberRudick, Victoria
dc.creatorMurray, Karen R.
dc.date.accessioned2019-08-22T21:14:52Z
dc.date.available2019-08-22T21:14:52Z
dc.date.issued2000-08-01
dc.date.submitted2013-10-23T07:50:06-07:00
dc.description.abstractMurray, Karen R., Immunoreactivity of Lecithin:Cholesterol Acyltransferase (LCAT); a Tool for Measurement of Levels and Characterization. Doctor of Philosophy (Biomedical Sciences), August, 2000, 162pp., 12 tables, 30 illustrations, bibliography, 150 titles. Lecithin:cholesterol acyltransferase is secreted by the liver into the plasma where it catalyzes the esterification of high density lipoprotein (HDL) cholesterol as part of the reverse cholesterol transport pathway. Via this pathway both HDL and LCAT have been linked to reducing the risk of atherosclerosis and coronary heart disease. These studies seek to develop an immunoassay to measure LCAT mass and to use immunoreactivity to elucidate the contribution of the highly conserved 121-136 domain of LCAT toward enzyme structure/function and HDL interaction. Several immunoassay models and antibody combinations were investigated to develop an ELISA assay for LCAT. Solid phase immunoassays were found to be most suitable for measuring LCAT from cell culture medium and in partially purified preparations. The immunoassay was analyzed for matrix interference, recovery studies, intra-run precision and inter-run precision. Evaluation of the immunoassay models and antibodies was extended to determine the potential for application toward measuring LCAT in plasma; however, the antibodies screened lacked the needed sensitivity. Studies were conducted to characterize the 121-136 region, a putative lipoprotein substrate binding domain. Differential immunoreactivity was demonstrated for a site directed antibody against the 121-136 region, in contrast to antibodies directed against the entire LCAT molecule, when the enzyme was bound to a hydrophobic surface or to substrate HDL, but not when bound to an alternate antibody. Three naturally occurring mutants within the 121-136 region, were tested for immunoreactivity with the same panel of antibodies and compared to wild type enzyme. These studies demonstrate that the 121-136 region of LCAT resides on the surface of the enzyme that has a high affinity for hydrophobic surfaces and mutation within this region significantly affects the exposure of different epitopes. This suggests that this region could plan an important role in enzyme interaction with its hydrophobic lipoprotein substrates and that mutations within this region could alter enzyme conformation affecting substrate interaction.
dc.format.mimetypeapplication/pdf
dc.identifier.urihttps://hdl.handle.net/20.500.12503/29187
dc.language.isoen
dc.provenance.legacyDownloads0
dc.subjectChemicals and Drugs
dc.subjectEnzymes and Coenzymes
dc.subjectLife Sciences
dc.subjectMedicine and Health Sciences
dc.subjectPhysiological Processes
dc.subjectLecithin cholesterol acyltransferase
dc.subjectLCAT
dc.subjecthigh density lipoprotein cholesterol
dc.subjectHDL cholesterol
dc.subjectimmunoassay
dc.subjecthydrophobic lipoprotein substrates
dc.titleImmunoreactivity of Lecithin: Cholesterol Acyltransferase (LCAT); A Tool for Measurement of Levels and Characterization
dc.typeDissertation
dc.type.materialtext
thesis.degree.departmentGraduate School of Biomedical Sciences
thesis.degree.disciplineBiomedical Sciences
thesis.degree.grantorUniversity of North Texas Health Science Center at Fort Worth
thesis.degree.nameDoctor of Philosophy

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