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The reaction of lecithin:cholesterol acyltransferase (LCAT) with high density lipoproteins (HDL) is of critical importance in reverse cholesterol transport. We studied the relationship between LCAT binding and HDL composition using two assays to determine LCAT binding affinity for discoidal reconstituted HDL (rHDL). We prepared rHDL with egg phosphatidylcholine (egg-PC), cholesterol, and either apolipoprotein (apo) A-I or apoA-II. The rHDL, identical in lipid content, had sizes of 96 and 100 A, respectively. Binding constants (Kd) determined by the activity-inhibition method were 2.2 +/- 0.3 x 10(-7) and 1.1 +/- 0.3 x 10(-16) M, whereas those determined by a solid-phase method were 3.3 +/- 0.9 x 10(-7) and 3.7 +/- 0.9 x 10(-7) M for apoA-I and apoA-II rHDL, respectively. The stoichiometry was 1 LCAT bound/rHDL. The appVmax/appKm for apoA-I was 80-fold higher than for apoA-II rHDL. The large difference between LCAT-binding constants and enzymatic activity measurements for the two particles suggests that LCAT binding and activation by apolipoproteins are independent events. To determine the effects of phospholipid headgroup on LCAT binding affinity, we tested rHDL containing up to 16 mol % of egg phosphatidylethanolamine (egg-PE), egg phosphatidic acid (egg-PA), or bovine phosphatidylserine (PS). Kd was unchanged for PS rHDL, but increased 6-fold with increasing PE content. AppVmax increased with PE content, but decreased with PS or PA relative to egg-PC controls. AppKm increased in PE rHDL, but remained unchanged in rHDL with PA or PS. Fluorescence characterization of the lipid domains of rHDL shows small differences in the polarity of the headgroup region of PE rHDL. Thus, LCAT binding is influenced by the lipid, but not the protein composition of rHDL. AppVmax values reflect active site preferences, while appKm values reflect interfacial binding affinity.