Activation of lecithin cholesterol acyltransferase by human apolipoprotein E in discoidal complexes with lipids.
In a continued investigation of lecithin cholesterol acyltransferase reaction with micellar discoidal complexes of phosphatidylcholine, cholesterol, and various water soluble apolipoproteins, we prepared complexes containing human apo-E by the cholate dialysis method. These complexes were systematically compared to apo-A-I complexes synthesized under the same reaction conditions. Apo-E complexes (134 A in diameter) were slightly larger than apo-A-I complexes (110 A) but were very similar in terms of their protein and lipid content (2.4:0.10:1.0, egg phosphatidylcholine/cholesterol/apolipoprotein, w/w) and in the percentage of apolipoprotein in alpha-helical structure (72-74%). Concentration and temperature-dependence experiments on the velocity of the lecithin cholesterol acyltransferase reaction revealed differences in apparent Km values and small differences in apparent Vmax but very similar activation energies (18-20 kcal/mol). These observations suggest that differences in lecithin cholesterol acyltransferase activation by apo-A-I and apo-E are primarily a result of different affinities of the enzyme for the particles but that the rate-limiting step of the reaction is comparable for both complexes. Apo-E was found to be 18% as effective as apo-A-I in activating purified human lecithin cholesterol acyltransferase. Addition of free apo-A-I to apo-E complexes resulted in the exchange of bound for free apolipoprotein causing a slight increase in the reactivity with the enzyme when the incubation mixture was assayed. When the unbound apolipoproteins were removed by ultracentrifugation reisolated complexes containing both apo-E and apo-A-I demonstrated an even greater increase in reactivity with the enzyme.
D016632:Apolipoprotein A-I; D001053:Apolipoproteins / Q000378:metabolism; D001054:Apolipoproteins A / Q000494:pharmacology; D001057:Apolipoproteins E / Q000494:pharmacology*; D002784:Cholesterol / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D008823:Micelles; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D013696:Temperature
D016632:Apolipoprotein A-I; D001053:Apolipoproteins; D001054:Apolipoproteins A; D001057:Apolipoproteins E; D008823:Micelles; D010713:Phosphatidylcholines; D002784:Cholesterol; D007862:Phosphatidylcholine-Sterol O-Acyltransferase