Binding of the partially purified glucocorticoid receptor of rat liver to chromatin and DNA

Partial purification of the activated glucocorticoid receptor of rat liver

“…Our procedure for the purification of the activated receptor takes advantage of this differencial affinity for phosphocellulose of the activated and the nonactivated (R-TA). The receptor-steroid complex is Incubations were performed at 20°C for 60 rain in 500 t~l assays containing 0.1 M NaCI and 0.1% BSA [4]. The amount of (R-TA) bound to chromatin (e e) and DNA (s m) was determined by centrifugation and washing of the pellets, followed by resuspension in Bray's solution [81 as described [4].…”

“…The receptor-steroid complex is Incubations were performed at 20°C for 60 rain in 500 t~l assays containing 0.1 M NaCI and 0.1% BSA [4]. The amount of (R-TA) bound to chromatin (e e) and DNA (s m) was determined by centrifugation and washing of the pellets, followed by resuspension in Bray's solution [81 as described [4]. first formed at low ionic strength and 0°C, in order to prevent activation of the (R-TA), and the cytosol is passed through two consecutive phosphocellulose columns, to which all proteins having affinity for the resine are bound.…”

“…Procedures for the preparation of rat liver chromatin and DNA°cellulose, as well as the conditions for the binding assay of the partially purified (R-TA) to this preparations have been reported in detail [4,9,10]. Determination of DNA was carried out by the procedure of Burton [11 ] and proteins were estimated with the technique of Lowry et al [12] scaled down to a sensitivity of 4-5 #g.…”

“…Studies on the mechanism of action of steroid hormones are presently concerned with the role of hormone receptors in enzyme induction and, more specifically, in the transcription of particular genes. The use of crude receptor preparations leads to artefactual results, due to the presence of nucleases and inhibitors which interfer with receptor binding to the nuclear acceptor sites [1][2][3][4]. Therefore, a prerequisite for this type of investigations is the availability of techniques for the purification of the receptor proteins.…”

Carrier‐free 8‐azidoadenosine 5′‐[γ‐32P]triphosphate

“…The low abundance of e.g. receptors in membranes, in the case for steroid hormones [6] or MSH [7] in the order of 10 4 per cell, requires that such photoaffinity label be of very high specific radioactivity.…”