title: Specific binding of free apolipoprotein A-I to a high-affinity binding site on HepG2 cells: characterization of two high-density lipoprotein sites.
abstract: In this paper, we present the first evidence that free apoA-I, without association with lipids, binds only to a high-affinity binding site (Kd = 1.8 microgram/mL, Bmax = 63.12 ng/mL). This is a new binding site of higher affinity (80-100 times) but of lower capacity than the binding sites already described for HDL. This is also the first evidence on HepG2 cells both of a high-affinity site (Kd = 0.685 microgram/mL, Bmax = 39.86 ng/mL) and of a low-affinity site (Kd = 55.65 micrograms/mL, Bmax = 665.45 ng/mL) for HDL. ApoA-I-DMPC complexes also present two binding components comparable to the HDL3 binding sites. This free apoA-I binding is specific, as shown by competition experiments, and allowed us to specifically study this high-affinity site, without interference of the low-affinity one. Kinetic rates of association/dissociation for the high-affinity site were faster than for the low-affinity site (10 and 20 min versus 40 and 30 min, respectively). The kinetic Kd values, derived from association and dissociation rate constants (Kd = 55.14 and 2.91 micrograms/mL), were of similar magnitude as the Kd values calculated by Scatchard analysis. These data confirm that HDL3 binding sites characterized by saturation experiments follow the law of mass action, indicative of ligand-receptor interaction. In summary, HepG2 cells present high HDL3 binding sites which are able to bind free apoA-I in contrast with the low-affinity HDL3 binding sites.
authors.affiliation: INSERM U 326, Hôpital Purpan, Toulouse, France.
authors.forename: R
authors.initials: R
authors.lastname: Barbaras
authors.forename: X
authors.initials: X
authors.lastname: Collet
authors.forename: H
authors.initials: H
authors.lastname: Chap
authors.forename: B
authors.initials: B
authors.lastname: Perret
mesh_terms: D016632:Apolipoprotein A-I / Q000378:metabolism*; D001667:Binding, Competitive; D006801:Humans; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL