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United States Department of Agriculture

Agricultural Research Service

Title: Selectivity of a C-Terminal Peptide Antiserum for Different Dna-Binding States of the Vitamin D Receptor

Authors
item Koszewski, N - UNIV KENTUCKY, LEXINGTON
item Reinhardt, Timothy
item Langub, M - UNIV KENTUCKY, LEXINGTON
item Malluche, H - UNIV KENTUCKY, LEXINGTON
item Horst, Ronald

Submitted to: Archives Of Biochemistry and Biophysics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 9, 1997
Publication Date: N/A

Interpretive Summary: Milk fever is a disease affecting 6-8% of all US dairy cows each year, which means approximately 700,000 cows are affected each year. This disease has been estimated to cost approximately $300/episode or $210 million annually as a result of treatment and production losses. The major clinical symptom seen in cows developing this disorder is their inability to stand and eventual lapse into coma if not treated medically. The cows lose muscle and nerve function because blood calcium concentrations fall below the level required to maintain normal electrical activity of the tissues. All mammals have evolved a very intricate system designed to maintain normal blood calcium. Calcium leaves the body in large amounts, when cows begin making milk which is rich in calcium. This calcium must be replaced, either by absorbing more from the diet or by removing calcium from the skeleton. In cows that develop milk fever, this system has broken down. Vitamin D plays a major role in controlling the movement of calcium into and out of the body by modifying a protein (vitamin D receptor). In this paper we described the development of an antibody against the vitamin D receptor. The antibody was used to study changes in physical state of the vitamin D receptor as influenced by the presence or absence of vitamin D, vitamin A and other controlling factors. This information will assist scientists, as well as nutritionists, and veterinarians alike in understanding how vitamin D is utilized by the body. The information may assist in developing various strategies for treating milk fever and other metabolic disorders in both human and veterinary medicine.

Technical Abstract: Antisera against peptides from the extreme N- and C-terminal regions of the VDR were evaluated. The N-terminal antiserum, Ab192, functioned as a general purpose antibody, being able to supershift the rhVDR in heterodimeric or homodimeric binding complexes in the EMSA, and detect both recombinant and native forms of the receptor on Western blots. The C-terminal antiserum, Ab195, also identified the receptor on Western blots, but in contrast, it displayed differential sensitivity to the conditions employed in the EMSA. In the presence of 1,25(OH)2D3, rhVDR, rhRXR-alpha and nonspecific DNA, Ab195 produced a weak supershift of the heterodimer complex in the EMSA. Significantly, omission of hormone from the binding buffer resulted in a complete shift of the bound complex with the antiserum. A complete supershift was also observed if only the nonspecific DNA was removed. Together these results indicate antiserum sensitivity to the ligand status in the rhVDR C-terminus as part of a DNA-bound heterodimer complex. Inclusion of 9-cis RA resulted in a modest increase in the amount of shifted product relative to 1,25(OH)2D3 alone. Finally, Ab195 completely supershifted the rhVDR homodimer binding complex under all tested conditions, including those analogous to where it was largely ineffective in shifting the heterodimer. Thus, Ab195 is sensitive to the ligand binding status of the VDR, discriminates heterodimer and homodimer binding interactions and should provide an additional tool for exploring conformational changes induced in the receptor.

Last Modified: 10/26/2014
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