Document Detail

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].
MedLine Citation:
PMID:  3768308     Owner:  NLM     Status:  MEDLINE    
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 data provide strong evidence that [3H]25-OH-D3-ANG was covalently linked to the rat DBP. This photoaffinity probe should provide a valuable tool for the analysis of the binding site on this transport protein.
R Ray; S A Holick; N Hanafin; M F Holick
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Biochemistry     Volume:  25     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  1986 Aug 
Date Detail:
Created Date:  1986-11-24     Completed Date:  1986-11-24     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  4729-33     Citation Subset:  IM    
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MeSH Terms
Affinity Labels / chemical synthesis*
Azides / chemical synthesis*,  metabolism
Binding, Competitive
Calcifediol / analogs & derivatives*,  chemical synthesis,  metabolism
Carbon Radioisotopes
Indicators and Reagents
Vitamin D-Binding Protein / blood*,  isolation & purification
Grant Support
Reg. No./Substance:
0/Affinity Labels; 0/Azides; 0/Carbon Radioisotopes; 0/Indicators and Reagents; 0/Vitamin D-Binding Protein; 10028-17-8/Tritium; 101396-04-7/25-hydroxyvitamin D3 3-(N-(4-azido-2-nitrophenyl)glycinate); 19356-17-3/Calcifediol

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