PMID: 3768308Aug 26, 1986Paper

Photoaffinity labeling of the rat plasma vitamin D binding protein with [26,27-3H]-25-hydroxyvitamin D3 3 beta-[N-(4-azido-2-nitrophenyl)glycinate

Biochemistry
R RayM F Holick

Abstract

It is well recognized that the vitamin D binding protein (DBP) is important for the transport of vitamin D, 25-hydroxyvitamin D (25-OH-D), and its metabolites. In an attempt to better understand the molecular-binding properties of this ubiquitous protein, we designed and synthesized a photoaffinity analogue of 25-OH-D3 and its radiolabeled counterpart. This analogue, 25-hydroxyvitamin D3 3 beta-[N-(4-azido-2-nitrophenyl)glycinate] (25-OH-D3-ANG), was recognized by the rat DBP and was about 10 times less active than 25-OH-D3 in terms of binding. Incubation of [3H]25-OH-D3 or [3H]25-OH-D3-ANG with rat DBP revealed that both compounds were specifically bound to a protein with a sedimentation coefficient of 4.1 S. Each was displaced with a 500-fold excess of 25-OH-D3. When [3H]25-OH-D3-ANG was exposed to UV radiation in the presence of rat DBP followed by the addition of a 500-fold excess of 25-OH-D3, there was no displacement of tritium from the 4.1S peak. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographic analysis of [3H]25-OH-D3-ANG exposed to UV radiation in the presence of rat DBP followed by the addition of a 500-fold excess of 25-OH-D3 revealed one major band with a molecular weight of 52 000. These d...Continue Reading

References

Jun 1, 1975·Proceedings of the National Academy of Sciences of the United States of America·S P DaigerL L Cavalli-Sforza
Jan 1, 1979·Annual Review of Biophysics and Bioengineering·M Das, C F Fox
Jan 1, 1977·Methods in Enzymology·H Bayley, J R Knowles
Jun 1, 1961·The American Journal of Physiology·D SCHACHTERH SCHENKER

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Citations

Mar 14, 2000·Bioorganic & Medicinal Chemistry Letters·N SwamyR Ray
Nov 13, 2013·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Victor Tuan Giam Chuang, Masaki Otagiri
Jun 1, 1997·Protein Expression and Purification·N SwamyR Ray

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