Open AccessKinetic analysis of transcellular passage of the cobalamin–transcobalamin complex in Caco-2 monolayers
Author(s) -
Christian B. Juul,
Sergey N. Fedosov,
Ebba Nexø,
Christian W. Heegaard
Publication year - 2019
Publication title -
molecular biology of the cell
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.463
H-Index - 225
eISSN - 1939-4586
pISSN - 1059-1524
DOI - 10.1091/mbc.e18-09-0571
Subject(s) - cobalamin , biology , transcytosis , receptor , transcellular , paracellular transport , caco 2 , ligand (biochemistry) , biophysics , biochemistry , intrinsic factor , microbiology and biotechnology , in vitro , endocytosis , membrane , vitamin b12 , permeability (electromagnetism)
We suggest a novel kinetic approach to quantifying receptor–ligand interactions via the cellular transport and/or accumulation of the ligand. The system of cobalamin (Cbl, vitamin B12) transport was used as a model, because Cbl is an obligatory cofactor, taken up by animal cells with the help of a transport protein and a membrane receptor. Bovine transcobalamin (bTC) stimulated the cellular accumulation and transcytosis of radioactive [ 57 Co]Cbl in polarized monolayers of Caco-2 cells. The bovine protein was much more efficient than human TC. The transport was inhibited in a dose-dependent manner by the unlabeled bTC-Cbl complex, the ligand-free bTC, and the receptor-associated protein (RAP). This inhibition pattern implied the presence of a megalin-like receptor. Quantitative assessment of kinetic records by the suggested method revealed the apparent concentration of receptors in vitro (≈15 nM), as well as the dissociation constants of bTC–Cbl ( K d = 13 nM) and RAP ( K d = 1.3 nM). The data were used to estimate the effective luminal concentrations of TC-specific receptors in kidneys (3.8 µM) and intestine (50 nM), the tissues resembling polarized Caco-2 cells.