Rate and equilibrium constants for binding of apo-E HDL c (a cholesterol-induced lipoprotein) and low density lipoproteins to human fibroblasts: Evidence for multiple receptor binding of apo-E HDL c

Abstract

Competitive binding assays have demonstrated that a cholesterol-induced canine lipoprotein containing only the E apoprotein (apo-E HDL_c) binds to the same cell surface receptors of human fibroblasts as human low density lipoproteins (LDL). However, the apo-E HDL_c have a much greater binding activity than LDL. Equilibrium and kinetic binding studies were conducted at 4°C to determine the mechanism for this enhanced receptor binding activity. Based on the data, the binding of both LDL and apo-E HDL_c appears to be a simple bimolecular receptor interaction, and no heterogeneity of binding sites or cooperative effects among the receptor sites were observed. Equilibrium dissociation constants determined by Scatchard analysis of the equilibrium binding data for apo-E HDL_c (K_d = 0.12 × 10^-9 M) and LDL (K_d = 2.8 × 10^-9 M) revealed a 23-fold greater affinity of HDL_c for the receptors. Association and dissociation rate constants for the lipoprotein-receptor complex were determined from the time course of binding at various lipoprotein concentrations. The equilibrium dissociation constants calculated from these kinetic data confirmed that apo-E HDL_c had a much higher affinity for the receptor than LDL. Furthermore, the kinetic studies indicated that apo-E HDL_c bound more rapidly than LDL with rates of association of 18.0 × 10⁴ and 5.5 × 10⁴ M^-1 sec^-1, respectively. The rate of dissociation of the apo-E HDL_c-receptor complex (1.7 × 10^-5 sec^-1) was slower than that of the LDL receptor complex (6.3 × 10^-5 sec^-1). An additional important difference between the binding of apo-E HDL_c and LDL was that 4 times (3.6 ± 0.4) as many LDL particles as HDL_c particles were required for saturation of the receptors at maximal binding. These data indicate that each HDL_c particle binds to multiple cell surface receptors at a ratio of 4:1 for LDL receptor binding.