Journal
MOLECULAR AND CELLULAR ENDOCRINOLOGY
Volume 328, Issue 1-2, Pages 56-62Publisher
ELSEVIER IRELAND LTD
DOI: 10.1016/j.mce.2010.07.002
Keywords
DHEA; Biotin; Benzophenone; Nitric oxide; Non-genomic
Categories
Funding
- American Heart Association
- Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development
- NIH [AG018928]
Ask authors/readers for more resources
To characterize the cell surface receptor for dehydroepiandrosterone (DHEA), we synthesized a DHEA analog containing biotin and benzophenone groups (DHEA-BP-Bt). DHEA-BP-Bt was equipotent with DHEA in competing with [H-3]DHEA for binding to solubilized plasma membranes of bovine aortic endothelial cells (BAEC). Additionally, DHEA-BP-Bt pre-conjugated to avidin and immobilized on agarose, also inhibited plasma membrane binding of [H-3]DHEA. Furthermore, DHEA-BP-Bt activated endothelial nitric oxide synthase, similar to DHEA. Confocal micrographs showed that, upon photoirradiation, DHEA-BP-Bt bound to sites on the cell surface of BAEC in a DHEA inhibitable manner. Finally, DHEA-BP-Bt bound specifically to proteins of approximately 55 kDa and 80 kDa, either when live cells were UV irradiated with the analog and plasma membrane proteins separated by SDS-PAGE or in a ligand blot analysis. These data confirm the successful synthesis of a photoactive, biotinylated DHEA analog which is capable of cross-linking to and identifying plasma membrane DHEA binding sites and which will allow us to further purify this receptor. (C) 2010 Elsevier Ireland Ltd. All rights reserved.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available