Open AccessRate 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
Author(s) -
Robert E. Pitas,
Thomas L. Innerarity,
Katrin Arnold,
Robert W. Mahley
Publication year - 1979
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.76.5.2311
Subject(s) - dissociation constant , ldl receptor , chemistry , receptor , lipoprotein , equilibrium constant , apolipoprotein b , high density lipoprotein , binding site , dissociation (chemistry) , low density lipoprotein , cholesterol , biophysics , biochemistry , biology
Competitive binding assays have demonstrated that a cholesterol-induced canine lipoprotein containing only the E apoprotein (apo-E HDLc ) binds to the same cell surface receptors of human fibroblasts as human low density lipoproteins (LDL). However, the apo-E HDLc 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 HDLc 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 HDLc (K d = 0.12 × 10-9 M) and LDL (K d = 2.8 × 10-9 M) revealed a 23-fold greater affinity of HDLc 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 HDLc had a much higher affinity for the receptor than LDL. Furthermore, the kinetic studies indicated that apo-E HDLc 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 HDLc -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 HDLc and LDL was that 4 times (3.6 ± 0.4) as many LDL particles as HDLc particles were required for saturation of the receptors at maximal binding. These data indicate that each HDLc particle binds to multiple cell surface receptors at a ratio of 4:1 for LDL receptor binding.