Kinetic analysis of transcellular passage of the cobalamin-transcobalamin complex in Caco-2 monolayers

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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 [ 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.

Original languageEnglish
JournalMolecular Biology of the Cell
Volume30
Issue4
Pages (from-to)467-477
Number of pages11
ISSN1059-1524
DOIs
Publication statusPublished - 15 Feb 2019

Keywords

  • Animals
  • Biological Transport
  • Caco-2 Cells
  • Cattle
  • Cobalt Radioisotopes
  • Humans
  • Kinetics
  • Ligands
  • Receptors, Cell Surface/metabolism
  • Transcobalamins/metabolism
  • Vitamin B 12/metabolism

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