Factors affecting the binding of lecithin:cholesterol acyltransferase with interfacial substrates
Bolin, Delmas John
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Permalink
https://hdl.handle.net/2142/20330
Description
Title
Factors affecting the binding of lecithin:cholesterol acyltransferase with interfacial substrates
Author(s)
Bolin, Delmas John
Issue Date
1994
Doctoral Committee Chair(s)
Jonas, Ana
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
The esterification of cholesterol on high density lipoproteins (HDL) catalyzed by lecithin:cholesterol acyltransferase (LCAT) maintains a gradient for cholesterol diffusion from cell membranes and atherosclerotic plaques and plays a critical part in the maintenance of cholesterol homeostasis. The principal protein of HDL, apolipoprotein A-I (apoA-I) is a major physiological activator of the LCAT reaction. Despite the importance of this reaction, the factors which influence LCAT affinity for the surface of HDL are poorly understood. To determine what properties of HDL influence LCAT binding affinity and reactivity, three sensitive methods for determining LCAT binding equilibrium with reconstituted HDL (rHDL) were developed: fluorescence energy transfer from DNS-LCAT to rHDL labeled with NBD-stearate; direct binding of $\sp{125}$I-LCAT to microtiter plates coated with rHDL; and an activity inhibition assay. Using these methods, LCAT binding affinity was demonstrated to be independent of apolipoprotein composition, indicating that activation of LCAT by apolipoproteins occurs at a separate reaction step from LCAT binding.
The effect of altered rHDL phospholipid composition on LCAT binding and reactivity was examined by adding up to 22mol% phosphatidylserine, phosphatidic acid, phosphatidylethanolamine, or sphingomyelin to rHDL prepared with bulk egg phosphatidylcholine, cholesterol, apoA-I. These rHDL particles had similar in sizes (97 A. Using the activity inhibition method, no change in LCAT binding affinity was observed for rHDL containing anionic phospholipids: however, increases in phosphatidylethanolamine increased the dissociation constant of LCAT from the rHDL. Increases in sphingomyelin also increased the dissociation constant, indicating decreased LCAT binding affinity. The apparent V$\sb{\rm max}$ of cholesterol esterification was decreased in rHDL containing anionic phospholipids, but was increased in rHDL containing phosphatidylethanolamine. These results suggest that the apparent V$\sb{\rm max}$ values reflect the acyl donor capacity of the phospholipids for the LCAT reaction. The apparent K$\sb{\rm m}$ was correlated with the dissociation constant, indicating that the apparent K$\sb{\rm m}$ reflects the binding affinity of LCAT with these substrates. Sphingomyelin content increased the apparent K$\sb{\rm m}$ but did not affect the apparent V$\sb{\rm max},$ demonstrating that sphingomyelin does not inhibit LCAT by competing for phosphatidylcholine binding at the active site.
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