SPECIFIC BINDING OF FREE APOLIPOPROTEIN-A-I TO A HIGH-AFFINITY BINDING-SITE ON HEPG2 CELLS - CHARACTERIZATION OF 2 HIGH-DENSITY-LIPOPROTEINSITES

In this paper, we present the first evidence that free apoA-I, without association with lipids, binds only to a high-affinity binding site ( K-d = 1.8 mu g/ML, B-MAX = 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 H epG2 cells both of a high-affinity site (Ka = 0.685 mu g/ML, B-MAX = 3 9.86 ng/ML) and of a low-affinity site (K-d 55.65 mu g/ML, B-MAX = 665 .45 ng/mL) for HDL. ApoA-I-DMPC complexes also present two binding com ponents comparable to the HDL(3) binding sites. This free apoA-I bindi ng is specific, as shown by competition experiments, and allowed us to specifically study this high-affinity site, without interference of t he 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 k(d) values, derived from association and dissociation rate constants (K-d = 55.14 abd 2.91 mu g/ML), were of similar magnitude as the K-d values calcula ted by Scatchard analysis. These data confirm that HDL(3) binding site s characterized by saturation experiments follow the law of mass actio n, indicative of ligand-receptor interaction. In summary, HepG2 cells present high HDL(3) binding sites which are able to bind free apoA-I i n contrast with the low-affinity HDL(3) binding sites.