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)

Journal Article · Tue Aug 26 00:00:00 EDT 1986 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00365a001· OSTI ID:6917356

Ray, R; Holick, S A; Hanafin, N; Holick, M F

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 (/sup 3/H)25-OH-D3 or (/sup 3/H)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 (/sup 3/H)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 (/sup 3/H)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 (/sup 3/H)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.

• Research Organization: Dept. of Agriculture/Human Nutrition Research Center on Aging, Boston, MA
• OSTI ID: 6917356
• Journal Information: Biochemistry; (United States), Vol. 17
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
PROTEINS
LABELLING
VITAMIN D
RECEPTORS
AZIDES
CHEMICAL BONDS
PHOTOCHEMICAL REACTIONS
RATS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ANIMALS
CHEMICAL REACTIONS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MAMMALS
MEMBRANE PROTEINS
NITROGEN COMPOUNDS
ORGANIC COMPOUNDS
RODENTS
VERTEBRATES
VITAMINS
550201* - Biochemistry- Tracer Techniques