The major goal of this research is to elucidate the substrate specificity and mechanism of action of lecithin cholesterol acyltransferase (LCAT), the enzyme responsible for the synthesis of extracellular cholesterol esters in human plasma. During the past grant period we prepared discoidal HDL analogs of various compositions and investigated their reactions with LCAT. We showed that all HDL apolipoproteins are capable of activating LCAT, but that a specific three-dimensional structure of apo A-I is required for optimal activation. In addition, we demonstrated that the enzyme activity is sensitive to the phosphatidylcholine (PG) chain length, unsaturation, and concentration in the interface, as well as to the anion composition of the medium. On the other hand, we determined that LCAT activity is not a function of the phase state of the lipids, of the cholesterol concentration, nor of the PC/cholesterol ratios in the substrate particles. We propose to investigate further the substrate selectivity and mechanism of the LCAT reaction by examining the interaction of the enzyme with various substrate particles, and determining the intrinsic kinetic constants from the kinetic and equilibrium binding results. This work will involve the preparation of milligram amounts of the enzyme, its fluorescence labeling, characterization of the enzyme structure in the presence and absence of various effectors, binding studies of LCAT with discoidal substrates, speherical HDL analogs of various sizes, and native subfractionated HDL. In addition, we will study the phospholipase reaction of LCAT with mixed detergent-lipid micelles, small PC micelles, and monomeric lipids and esters, in order to gain a better understanding of the catalytic step, and the interface and activator apolipoprotein roles in the enzymatic reaction.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL029939-06
Application #
3340991
Study Section
Metabolism Study Section (MET)
Project Start
1983-07-01
Project End
1991-06-30
Budget Start
1988-07-01
Budget End
1989-06-30
Support Year
6
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Illinois Urbana-Champaign
Department
Type
Schools of Arts and Sciences
DUNS #
041544081
City
Champaign
State
IL
Country
United States
Zip Code
61820
Kosman, J; Jonas, A (2001) Deletion of specific glycan chains affects differentially the stability, local structures, and activity of lecithin-cholesterol acyltransferase. J Biol Chem 276:37230-6
Cho, K H; Durbin, D M; Jonas, A (2001) Role of individual amino acids of apolipoprotein A-I in the activation of lecithin:cholesterol acyltransferase and in HDL rearrangements. J Lipid Res 42:379-89
Jonas, A (2000) Lecithin cholesterol acyltransferase. Biochim Biophys Acta 1529:245-56
Cho, K H; Jonas, A (2000) A key point mutation (V156E) affects the structure and functions of human apolipoprotein A-I. J Biol Chem 275:26821-7
Jin, L; Shieh, J J; Grabbe, E et al. (1999) Surface plasmon resonance biosensor studies of human wild-type and mutant lecithin cholesterol acyltransferase interactions with lipoproteins. Biochemistry 38:15659-65
Kosek, A B; Durbin, D; Jonas, A (1999) Binding affinity and reactivity of lecithin cholesterol acyltransferase with native lipoproteins. Biochem Biophys Res Commun 258:548-51
Adimoolam, S; Jin, L; Grabbe, E et al. (1998) Structural and functional properties of two mutants of lecithin-cholesterol acyltransferase (T123I and N228K). J Biol Chem 273:32561-7
Adimoolam, S; Lee, Y P; Jonas, A (1998) Mutagenesis of highly conserved histidines in lecithincholesterol acyltransferase: identification of an essential histidine (His 377). Biochem Biophys Res Commun 243:337-41
Jonas, A (1998) Regulation of lecithin cholesterol acyltransferase activity. Prog Lipid Res 37:209-34
Lee, Y P; Adimoolam, S; Liu, M et al. (1997) Analysis of human lecithin-cholesterol acyltransferase activity by carboxyl-terminal truncation. Biochim Biophys Acta 1344:250-61

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