Functional Characterization and Purification of an Intracellular Vitamin D-binding Protein in Vitamin D-resistant New World Primate Cells
| Autor: | John S. Adams, Thomas R. Lebon, Mercedes A. Gacad, Hong Chen, Jonathan E. Arbelle |
|---|---|
| Rok vydání: | 1997 |
| Předmět: |
Gel electrophoresis
chemistry.chemical_classification Vitamin Vitamin D-binding protein medicine.medical_treatment Peptide Cell Biology Biology Ligand (biochemistry) Biochemistry Molecular biology Steroid chemistry.chemical_compound chemistry Vitamin D and neurology medicine Molecular Biology Intracellular |
| Zdroj: | Journal of Biological Chemistry. 272:8433-8440 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.272.13.8433 |
| Popis: | Most genera of New World primates exhibit resistance to vitamin D. These monkeys harbor high circulating concentrations of the prohormone 25-hydroxyvitamin D and the active vitamin D hormone 1,25-dihydroxyvitamin D. Previous work from this laboratory indicated that resistance is associated with the overexpression of a 60-65-kDa intracellular protein that binds vitamin D metabolites competitively. In the current studies 25-[3H]hydroxyvitamin D3 (25-OHD3) was used as a competitive ligand to investigate the ability of a number of small lipid molecules to interact with this intracellular vitamin D-binding protein (IDBP) in post-nuclear extracts of a prototypical lymphoblast cell line from the common marmoset, a vitamin D-resistant New World primate. Only those vitamin D metabolites with a hydroxyl moiety in the C-25 position were bound by IDBP. Disruption of the C-25 hydroxyl obviated binding, whereas more proximal alterations in the vitamin D side chain did not. Modifications in the A-ring of 25-hydroxylated vitamin D metabolites, most specifically hydroxylation of C-1, diminished but did not abolish ligand binding. Of more than two dozen other small lipid molecules examined, only the C-19 17-hydroxysteroids, 17β-estradiol and testosterone, and the C-21 steroid progesterone were found to be capable of binding specifically to IDBP. Using a combination of physical and serial chromatographic techniques, we enriched IDBP 25-OHD3 binding activity 17,588-fold in extracts of B95-8 cells. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this purified fraction demonstrated a predominant 65-kDa molecular species with a pI ~ 4.5. Seven different peptide fragments were isolated from the 65-kDa protein, each possessing sequence similarity to the hsp-70 family of proteins. Ligand binding analyses confirmed that human inducibly expressed hsp-70-bound 25-OHD3 with approximately similar affinity (~10−7 M) as did purified IDBP. In summary, these results suggest a novel action for the hsp-70 family of proteins as intracellular vitamin D- and gonadal steroid hormone-binding molecules. |
| Databáze: | OpenAIRE |
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