Affinity labeling of rat serum vitamin D binding protein.
Vitamin D binding protein (DBP) plays an essential role in the vitamin D hormone endocrine system in sequestering vitamin D3 and its metabolites with high affinity, and transporting them to various target organs and tissues. In the present investigation, 25-hydroxyvitamin D3-3 beta-(1,2-epoxypropyl)ether (25-OH-D3-epoxide) and 25-hydroxyvitamin D3-3 beta-bromoacetate (25-OH-D3-BE), synthetic analogs of 25-hydroxyvitamin D3 (25-OH-D3), were developed as affinity alkylating reagents for the covalent modification of the 25-OH-D3-binding site in rat vitamin D binding protein (rDBP). Competitive radioligand binding assays of 25-OH-D3-BE and 25-OH-D3-epoxide with affinity-purified rDBP demonstrated that these analogs displaced 25-hydroxy[26(27)-3H]vitamin D3 (3H-25-OH-D3), specifically bound to rDBP, in a dose-dependent fashion. Incubation of rDBP samples with radiolabeled versions of these analogs, i.e., 3H-25-OH-D3-epoxide and 3H-25-OH-D3-BE, resulted in the covalent labeling of rDBP. This labeling was largely prevented when incubations were carried out in the presence of an excess of 25-OH-D3, the natural ligand for rDBP. Labeling-specificity by these analogs was further demonstrated by the covalent labeling, inhibited by coincubation with a large excess of 25-OH-D3, of a single protein band, upon incubating rat serum Cohn IV fraction with 3H-25-OH-D3-epoxide and 3H-25-OH-D3-BE. Collectively, these results strongly suggested that 3H-25-OH-D3-epoxide and 3H-25-OH-D3-BE covalently modified the 25-OH-D3-binding site in rDBP. The reagents described in this report could be important in mapping the 25-OH-D3-binding pocket in rDBP.
Swamy
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Department of Medicine, Boston University School of Medicine, Massachusetts, USA.
Ray
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D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002112:Calcifediol / Q000031:analogs & derivatives / Q000378:metabolism; D002762:Cholecalciferol / Q000031:analogs & derivatives; D066298:In Vitro Techniques; D011869:Radioligand Assay; D051381:Rats; D014809:Vitamin D-Binding Protein / Q000097:blood* / Q000737:chemistry / Q000378:metabolism
C094126:25-hydroxyvitamin D3-3-(1,2-epoxypropyl)ether; C101470:25-hydroxyvitamin D3-bromoacetate; D000345:Affinity Labels; D014809:Vitamin D-Binding Protein; D002762:Cholecalciferol; D002112:Calcifediol