Date of Award

8-1-2000

Degree Type

Restricted Access Dissertation

Degree Name

Doctor of Philosophy

Field of Study

Biomedical Sciences

Department

Graduate School of Biomedical Sciences

First Advisor

Andras G. Lacko

Second Advisor

Victoria Rudick

Abstract

Murray, 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.

Comments

Murray, 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. W 4 M982I 2000

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