LCAT activation properties of apo A-I CNBr fragments and conversion of discoidal complexes into spherical particles.
We studied the substrate properties of the phospholipid-cholesterol-apolipoprotein complexes generated with apo A-I, apo A-I-CNBr fragments, apo A-II and apo A-IV for cholesterol esterification by the enzyme lecithin-cholesterol acyltransferase (LCAT). The kinetic parameters determined with the different complexes as substrates, showed that the complexes containing apo A-I and apo A-IV were about 40-times more efficient than those generated with the apo A-I fragments. In this system, the substrates containing apo A-II had the lowest efficiency. In spite of the differences in the kinetic parameters observed with the various apolipoprotein-lipid complexes, the cholesterol inserted in the complexes was esterified for more than 90% after 24 h in all systems studied. Based upon the results of the kinetic experiments, we followed the transformation of the discoidal complexes into spherical particles, due to the formation of a cholesteryl esters core, in the presence of low-density lipoproteins as an external source of cholesterol. We observed the formation of spherical particles by electron microscopy, after incubation of the discoidal complexes with LCAT for 24 h. The average percentage of cholesteryl esters in the converted particles was around 60% of the total cholesterol, varying between 40% for the apo A-I-CNBr-1-DPPC-cholesterol complex and up to 86% for the apo A-I-DPPC-cholesterol complex. The secondary structure of protein in the complexes was not significantly modified. However, the phospholipid phase transition disappeared, together with the parallel orientation of the phospholipid acyl chains with the helical segments of the apolipoproteins, as the phospholipids are organized in a monolayer at the surface of the spheres.
Vanloo
B
B
A.Z. St.-Jan, Department of Clinical Chemistry, Brugge, Belgium.
Taveirne
J
J
Baert
J
J
Lorent
G
G
Lins
L
L
Ruyschaert
J M
JM
Rosseneu
M
M
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000737:chemistry; D016632:Apolipoprotein A-I / Q000737:chemistry / Q000494:pharmacology*; D016633:Apolipoprotein A-II / Q000494:pharmacology; D001054:Apolipoproteins A / Q000494:pharmacology; D003488:Cyanogen Bromide; D004789:Enzyme Activation / Q000187:drug effects; D005454:Fluorescence Polarization; D007700:Kinetics; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000737:chemistry; D013329:Structure-Activity Relationship; D013696:Temperature
D016632:Apolipoprotein A-I; D016633:Apolipoprotein A-II; D001054:Apolipoproteins A; D010713:Phosphatidylcholines; C022205:apolipoprotein A-IV; D015060:1,2-Dipalmitoylphosphatidylcholine; C055402:1-palmitoyl-2-linoleoylphosphatidylcholine; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; D003488:Cyanogen Bromide