Kinetics of low-density lipoprotein receptor activity in Hep-G2 cells: derivation and validation of a Briggs-Haldane-based kinetic model for evaluating receptor-mediated endocytotic processes in which receptors recycle.
The process of receptor-mediated endocytosis for receptors that recycle to the cell surface in an active form can be considered as being kinetically analogous to that of a uni-substrate, uni-product enzyme-catalysed reaction. In this study we have derived steady-state initial-velocity rate equations for this process, based on classical Briggs-Haldane and King-Altman kinetic approaches to multi-step reactions, and have evaluated this kinetic paradigm, using as a model system the low-density lipoprotein (LDL)-receptor-mediated endocytosis of the trapped label [14C]sucrose-LDL in uninduced, steady-state Hep-G2 cells. Using the derived rate equations, together with experimentally determined values for Bmax (123 fmol/mg of cell protein), Kd (14.3 nM), the endocytotic rate constant ke (analogous to kcat; 0.163 min-1), Km (80 nM) and maximal internalization velocity (26.4 fmol/min per mg), we have calculated the ratio ke/Km (0.00204 nM-1.min-1), the bimolecular rate constant for LDL and LDL-receptor association (0. 00248 nM-1.min-1), the first-order rate constant for LDL-LDL-receptor complex dissociation (0.0354 min-1), the total cellular content of LDL receptors (154 fmol/mg of cell protein), the intracellular LDL receptor concentration (30.7 fmol/mg of cell protein) and the pseudo-first-order rate constant for LDL receptor recycling (0.0653 min-1). Based on this mathematical model, the kinetic mechanism for the receptor-mediated endocytosis of [14C]sucrose-LDL by steady-state Hep-G2 cells is one of constitutive endocytosis via independent internalization sites that follows steady-state Briggs-Haldane kinetics, such that LDL-LDL-receptor interactions are characterized by a rapid-high-affinity ligand-receptor association, followed by ligand-receptor complex internalization that is rapid relative to complex dissociation, and by receptor recycling that is more rapid than complex internalization and that serves to maintain 80% of cellular LDL receptors on the cell surface in the steady-state. The consistency with which these quantitative observations parallel previous qualitative observations regarding LDL-receptor-mediated endocytosis, together with the high correlation between theoretical internalization velocities (calculated from determined rate constants) and experimental internalization velocities, underscore the validity of considering receptor-mediated endocytotic processes for recycling receptors in catalytic terms.
Harwood
H J
HJ
Department of Metabolic Diseases, Pfizer Central Research, Pfizer Inc., Eastern Point Road, Groton, CT 06340, USA.
Pellarin
L D
LD
D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology*; D004705:Endocytosis; D006801:Humans; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology*; D008954:Models, Biological; D009363:Neoplasm Proteins / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D011973:Receptors, LDL / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D008077:Lipoproteins, LDL; D009363:Neoplasm Proteins; D011956:Receptors, Cell Surface; D011973:Receptors, LDL