期刊
STEROIDS
卷 142, 期 -, 页码 2-5出版社
ELSEVIER SCIENCE INC
DOI: 10.1016/j.steroids.2017.09.004
关键词
Corticosteroid; Nongenomic; Denaturation; Dialysis; NMR; Steroid
资金
- NIH [2R01 MH066958, R01 MH104373]
Cellular effects of glucocorticoids can be separated into classical transcriptional regulation via activation of the canonical nuclear glucocorticoid receptor and rapid actions mediated by activation of one or more putative membrane-associated glucocorticoid receptors that regulate both transcriptional and non-transcriptional signaling. Dexamethasone-bovine serum albumin (Dex-BSA) is one of several membrane-limited steroid receptor agonists. Dex-BSA and other steroid conjugates such as corticosterone-, estradiol- and testosterone-BSA have been used to study rapid steroid effects initiated by putative membrane receptors. The purity and stability of the steroid-BSA conjugate is crucial, therefore, since any steroid that is not bound to or that dissociates from the BSA conjugate could penetrate into the intracellular compartment and confound the experiment. We used fluorine NMR to determine if free Dex could be detected in a commercially available Dex-BSA dissolved in H2O. Non-covalently bound Dex was detected in the Dex-BSA solution, but the level of free Dex remained constant over time and with increasing temperature, indicating that the free Dex was not a result of instability of the Dex-BSA conjugate. The free Dex was lost when the Dex-BSA was denatured and subjected to dialysis, which suggested that it was trapped in the Dex-BSA three-dimensional structure and not covalently bound to the BSA. The purified, renatured Dex-BSA retained its rapid activity, which confirmed that the observed effects of Dex-BSA are not caused by non-covalently-bound Dex. Therefore, the Dex contaminant found in the Dex-BSA solution is likely to be tightly, but non-covalently, bound to BSA, and the Dex-BSA activity remains membrane-limited. Our findings indicate that Dex-BSA remains a suitable membrane-restricted glucocorticoid receptor agonist, but suggest that denaturing purification is a useful control for the study of membrane-initiated steroid-BSA actions.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据