Purification And Assay Of Glucocorticoid Receptor In The Rat Liver Cytosol With Hplc

Glucocorticoid (GC) has been widely used now and it binds to glucocorticoid receptor(GR) to produce its biological effect. CR is a member of the steroid receptor superfamily and lies in the nuclear. Like other steroid receptors, the feature of GR is high-affinity, low binding capacity and high specificity in binding to CC. But in clinical application we find that high dose CC is necessary in stress and in the treatment of shock and severe inflaximiatioit Until now its molecular mechanism is not clear. Professor Xu Renbao suggested that CC binds to high-affinity glucocorticoid receptor in physical state and in pathologic state it binds to low-affinity glucocorticoid receptor. Le Yingying, et al prove that low-affinity glucocorticoid receptor is existant in the liver of the rat with radiative ligand binding assay. However, CC must be studied as an entity to prove this hypothesis. The object of this study is to obtain the purified CR of the rat liver with IIPLC as well as radioligand binding assay , to prove the pupurified CR with Western blot and to probe to assay CR with HPLC. The following is done in our study to purify and assay GR 1. The purification of CR in rat liver with HPLC.high performance hydrophobic interaction chromatography(1-IPFIIC),Synchropak Propyl column and anti? phase elution procedure were applied in our study.Because the method of ultraviolet assay is not sensetive enough to valuate GR ,we measure it with isotopeabelling method. The cytosol of rat liver, binded with [3H]Dexamethasone(Dex),was performed with IIPLC at various temperature and various concentration of ligand. Elutions were collected every two minutes and radioactivity was assayed. At 0? and the concentration of [3H]Dex is lower than SOnmol/L,GR was eluted at 9lminutes; However,at 25 0C and the concentration of Ii3H]Dex is higher than SOnmol/L, two peaks appear at 3? and 9?1 minutes.That is to say that two molecules of GR which can bind to the ligand is existant. 2. The identification of GR of rat liver. Supernatant of cytosol of rat liver was performed with FIPLC and collected at 3?, 5?, 7?, 9?1 minutes again and again. After dialysis and dehydration, the concentrated GR was qualitative analysed with SDSAGE and WESTERN BLOT. SDSAGE showed that several bands appeared at the elutions of 3? mitntes and 5? minutes, no obvious bands appeared at the elutions of 7? minutes and a clear band of 60 Kd appeared at the elution of 9lminutes. WESTERN BLOT showed that there were positive bands of 60 Kd at the elutions of 5? minutes and 9lminutes and there were not any band at the elution of 3? minutes and 7? minutes, which suggested that there were GR in the elution of 5? and 9?1 minutes which can bind to the antibody of GR.3. the assay of GR of rat liver with areC.Traditional method of radiative 1igand--binding assay needs largeamunt of smp1e, high exrmse and isotoPC. We tried to assay GR withHPLC. SffiWGE suggested that a c1ear band of 60 Kd apPeared at thee1ution of 9--11 minutes and it is proved to be GR with WESTERN BL0T.the cytosol of rat 1iver was di1uted to various concentrations andperformed with HPLC. the elution of 9--11 minutes was co11ected andprotein concentration was measured with Bradford method. It is foUndthat protein concentrations at the range of 2th120Ing/L was lined withA595.