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  • Title: Kinetic analysis of transcellular passage of the cobalamin-transcobalamin complex in Caco-2 monolayers.
    Author: Juul CB, Fedosov SN, Nexo E, Heegaard CW.
    Journal: Mol Biol Cell; 2019 Feb 15; 30(4):467-477. PubMed ID: 30565973.
    Abstract:
    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 [57Co]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 ( Kd = 13 nM) and RAP ( Kd = 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.
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