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Abstract
The lecithin:cholesterol acyltransferase (LCAT) kinetics and activation energy and the stability of apolipoprotein A-I (apoA-I) were investigated using recombinant HDL (rHDL) containing phosphatidylcholine (PC), [3H]cholesterol, and apo A-I. The PC component of the rHDL contained sn-1 16:0 and sn-2 18:1 (POPC), 20:4 (PAPC), 20:5 n-3 (PEPC), or 22:6 n-3 (PDPC) or 10% of the respective PC species and 90% sn-1 18:1, sn-2 16:0 PC ether (OPPC ether). The appVmax of the rHDL containing 100% PC varied 10-fold and was ordered POPC > PEPC > PAPC > PDPC, whereas the appKm values varied 19-43 microns PC. The ether-containing rHDL had appVmax values 17-40% of their respective 100% PC rHDL, but maintained the same rank order. The activation energy of LCAT was lower for rHDL containing long chain polyunsaturated fatty acids (PUFA) compared to rHDL containing 100% POPC or 10% PC/90% OPPC ether. The concentration of guanidine HCl (D1/2) required to denature one-half of the apoA-I on rHDL containing long chain PUFA was reduced (1.2-16 M) compared to those containing 100% POPC or 10% PC/90% OPPC (2.2-2.4 M) and there was a strong correlation (r = 0.71) between LCAT activation energy and the stability of apoA-I (i.e., D1/2). We conclude that long chain PUFA in the sn-2 position of PC decreases the catalytic efficiency of LCAT, the activation energy of the LCAT reaction and the stability of apoA-I on the rHDL particles. The strong association between rHDL apoA-I stability and LCAT activation energy suggests that the temperature-dependent step of the LCAT reaction may be sensitive to the strength of the interaction of apoA-I with rHDL PC.
Highlights
The lecithin: cholesterol acyltransferase (LCAT) kinetics and activation energy and the stability of apolipoprotein A-I were investigated using recombinant high density lipoproteins (HDL) containing phosphatidylcholine (PC), ['H]cholesterol, and apo A-I
Stokes' radius was determined by electrophoresis using 4-30% polyacrylamide gradient gels. recombinant HDL (rHDL) containing 10% PC/90% OPPC ether were reisolated by HPLC on a Superose 12B column as described in the Methods section
Using rHDL of comparable size and composition containing defined PC species or containing an interface of 90% OPPC ether and 10%of the defined PC species, we investigated the reaction kinetics of cholesterol esters (CE) formation by LCAT and the activation energy and the stability of apolipoprotein A-I (apoA-I) on rHDL
Summary
The lecithin: cholesterol acyltransferase (LCAT) kinetics and activation energy and the stability of apolipoprotein A-I (apoA-I) were investigated using recombinant HDL (rHDL) containing phosphatidylcholine (PC), ['H]cholesterol, and apo A-I. The activation energy of LCAT was lower for rHDI, containing long chain polyunsaturated fatty acids (PUFA) compared to rHDL containing 100% POPC or 10% PC/90% OPPC ether. The concentration of guanidine HCI (D1,2)required to denature onehalf' of the apoA-I on rHDL containing long chain PUFA was reduced ( 1 2-1.6 M ) compared to those containing 100% POPC or 10% PC/90% OPPC (2.2-2.4 M ) and there was a strong correlation ( P = 0.71) between LCAT activation energy and the stability of apoA-I Long-chain polyunsaturated fatty acids in the sa position of phosphatidylcholine decrease the stability of recombinant high density lipoprotein apolipoprotein AI and the activation energy of the 1ecithin:cholesterol acyltransferase reaction.
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