Reaction of lecithin:cholesterol acyltransferase with micellar complexes of apolipoprotein A-I and phosphatidylcholine, containing variable amounts of cholesterol.
In a continued investigation of lecithin:cholesterol acyltransferase reaction with micellar, discoidal complexes of phosphatidylcholine (PC) . cholesterol . apolipoprotein A-I (apo-A-I), we prepared well defined complexes with variable free cholesterol contents and examined their reactivity with purified enzyme. The complexes, prepared by the sodium cholate dialysis method, were fractionated into "small" and "large" classes by gel filtration of the reaction mixtures through a Bio-Gel A-5m column. The small complexes had egg-PC/cholesterol/apo-A-I molar ratios from 68:14:1 to 80:1:1, discoidal shapes with diameters around 114 (+/- 13) A and widths of 42 A by electron microscopy, and Stokes radii from 47 to 49 A corresponding to molecular weights near 2 X 10(5). The corresponding properties of the large complexes, isolated from samples with higher cholesterol contents, were egg-PC/cholesterol/apo-A-I molar ratios from 84:26:1 to 96:17:1, diameters of 161 (+/- 20) A, widths of 43 A, Stokes radii around 80 A, and estimated molecular weights in the vicinity of 5 X 10(5). Both types of complexes, when adjusted to equal apo-A-I concentrations, gave essentially identical initial reaction velocities with purified lecithin:cholesterol acyltransferase over a wide range of cholesterol concentrations (from 2 X 10(-7) to 4 X 10(-4) M), PC/cholesterol molar ratios (from 3:1 to 12:1), and quite different lipid fluidity conditions as detected by diphenylhexatriene fluorescence polarization. When complexes were adjusted to a constant cholesterol concentration, the initial velocities of the lecithin:cholesterol acyltransferase reaction followed Michaelis-Menten kinetics relative to the apo-A-I concentrations. Arrhenius plots of initial reaction rates for various complexes with variable cholesterol content and fluidity, measured at constant apo-A-I concentrations, gave identical temperature dependences with an average activation energy of 18.0 kcal/mol. These results strongly suggest that the cholesterol esterification on high density lipoprotein particles does not depend on their unesterified-cholesterol contents, PC/unesterified-cholesterol molar ratios, nor on the fluidity of their lipid domains.
D016632:Apolipoprotein A-I; D001053:Apolipoproteins / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D002850:Chromatography, Gel; D003102:Colloids; D003956:Dialysis; D005454:Fluorescence Polarization; D008823:Micelles; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism*
D016632:Apolipoprotein A-I; D001053:Apolipoproteins; D003102:Colloids; D008823:Micelles; D010713:Phosphatidylcholines; D002784:Cholesterol; D007862:Phosphatidylcholine-Sterol O-Acyltransferase