Chromatographic separation on phosphocellulose of activated and nonactivated forms of steroid-receptor complex. Purification of the activated complex

The 90 000-M, glucocorticoid receptor was purified to homogeneity according to sodium dodecylsulfate/polyacrylamide gel electrophoresis. An affinity column containing either dexamethasone-17P-carboxylic acid or dexamethasone-21 -methanesulfonate bound to the matrix with the help of a disulfide bond is used in this study. Using this affinity matrix, in a single step, 8700-fold purification of glucocorticoid receptor from rat liver cytosol could be achieved. Following the method of activation and DNA-cellulose chromatography the 90000-M, receptor subunit was purified to homogeneity.The existence of steroid binding proteins in the target organ has been investigated in many laboratories [1 -71. The glucocorticoid resceptor protein from rat liver cytosol has been characterised in crude or partially purified receptor preparations [8 -191. The partial purification of the glucocorticoid receptor by a two-step chromatographic procedure on either phosphocellulose [20,21] or DNA-cellulose [19] with an intermediate activation step has been reported by several groups. We report here the purification of glucocorticoid receptor subunit of molecular weight 90 000 to homogeneity by hormone-affinity chromatography, activation and DNA-affinity chromatography. MATERIALS AND METHODSCytaminium dihydrochloride (Merck, Darmstadt), dexamethasone from Sigma Chemical Co, [3H]dexamethasone (2.5 Ci/mmol) from Amersham Buchler (Braunschweig). All other chemicals were of analytical grade obtained commercially. BuffersBuffer A: 1 mM EDTA, 20 mM sodium phosphate, 10 glycerine, 50 mM KC1, pH 7.0; buffer B: 1 mM EDTA, 20 mM sodium phosphate, 10 % glycerine, 2 mM 2-niercaptoethanol, 90 mM KCl, pH 7.8.Oxidution of 9a-Fluoro-16sr-methylprednisolone u d h Periodic Acid (221 1 g dexamethasone (spec.act. 1 mCi/g) was dissolved in 200 ml ethyl alcohol. 120 ml (0.025 M) periodic acid and 3.8 ml (2.5 M) sulfuric acid in 75 ml distilled water were added to this solution. After stirring for 24 h at room temperature, alcohol was removed under reduced pressure, the precipitated crude product was collected by filtration and dried over phosphorous pentoxide. The crude acid thus obtained was dissolved in 1 1 ethyl acetate and washed 3 times with 100-ml portions of 0.01 M NaOH. The wash was brought to pH 2 with 1 M HCl; kept for 2 h at room temperature and stored at 4 "C overnight. The acid was collected by filtration and recrystallised from methanol/water to yield pure product (yield 945 mg, 98%). m.p. > 250°C; mass spectrum (high resolution) m/e found 378.1838, calculated 378.1842. NMR 6 = 115 ppm, 1.6ppm (C-18 and C-19), 0.92ppm doublet) 16a-methyl; RF = 0.048 (CHC13/MeOH, 9 : l), RF = 0.58 (ethyl acetate/ethanol/NH3, 5 : 5 : 1). Synthesis o j Dex-I7~-curhoxy-his-~-aminoetkanethiol0.5 mmol (189 mg, spec.act. 1 pCi/mg) dex-17P-carboxylic acid was added to a solution of N-hydroxysuccinimide (57.5 mg, 0.5 mmol) in dry ethyl acetate (8 ml). A solution of dicyclohexylcarbodiimide (103 mg, 0.5 mmol) in 2 ml dry ethyl acetate was added and the reaction mixture was le...

Section: Discussionmentioning

confidence: 99%

Three‐Step Purification of Glucocorticoid Receptors from Rat Liver

Govindan

Manz

1980

“…Inorganic ions may accelerate or slow down both activation and deactivation and this has led to controversy in the literature [4,5]. Single-time-point assays did not allow detection of activation in the presence of CaZf or molybdate ions [I 2,161.…”

Section: Discussionmentioning

confidence: 99%

Activation and Deactivation of the Glucocorticoid Hormone‐Receptor Complex

Arányi¹

1983

Cytosol glucocorticoid receptors acquired the ability to bind to DNA-cellulose on incubation at 25 -37' C and/or in media of high ionic strength (0.3 M KCI). However, this activation was transient only. It was followed by deactivation whose rate was also dependent on incubation temperature and on the presence of potassium chloride. Deactivation resulted in a decreased but non-zero binding to DNA-cellulose. Specific triamcinolone acetonide binding to the receptor protein was not lost under the same conditions. Deactivation commenced before it became apparent, probably together with activation. Preactivated complex underwent deactivation even in conditions that would not allow significant decrease in DNA-cellulose binding without previous incubation at 37 "C.In contrast to previous reports it was found that fast activation/deactivation took place in the presence of 4 m M C a z f .Molybdate ions slow down both activation and deactivation, but do not prevent activation by heat.Heat activatedldeactivated complex differed in size from both non-activated and Ca2+-deactivated complexes. This finding suggests that heat and Ca2+-induced deactivation follow different mechanisms.The chain of events that leads to the expression of glucocorticoid hormone action starts with the formation of a complex between the steroid and its receptor protein in the cytoplasm of the target cell. The hormone-receptor complex then undergoes a process called activation that renders it capable of nuclear binding. (For a review see [I].) Activation can be studied in vitro in cell-free systems [2-41.The activated complex is similar in size to but differs in charge distribution from its non-activated counterparts [5]. Activation can be promoted by elevated temperature and/or ionic strength, addition of ADP or by removal of low-molecular-weight inhibitor(s) [2,5,7-91. The process was claimed to reach equilibrium, whose position could be influenced by pH and temperature [lo, 1 I]. Inorganic ions such as molybdate prevented activation [12,13], and binding of the activated complex to nuclei or D N A was inhibited by chemical modification by pyridoxal phosphate [14,15]. Contradictory reports concerning the role of Ca2+ ions have been published [2,5,16,17].In spite of the considerable attention paid to activation, our knowledge about the physicochemical changes that occur during its course is rather limited.It has been observed that in certain conditions the activated complex lost its DNA-binding properties without releasing the bound hormone, i.e. the activated complex was not the end-product of the cytoplasmic processing, rather activation was followed by deactivation [lo]. Nothing is known, however, about the details of the deactivation step. We undertook to study deactivation of the glucocortocoidhormone -receptor complex in the hope of shedding a little more light on the intricate mechanism of activation, as well. ~~~Ahbrevialions. TA, triamcinolone acetonide; TA-R, triamcinoloneacetonide -receptor complex.

“…Both receptors [22] and casein kinases [23] are known to bind to phosphocellulose, but the latter are eluted at higher salt concentration than the former. Chromatography of immunoaffinity purified receptor (Fig.…”

Section: Separation Of the Receptor From The Kinase Activitymentioning

confidence: 99%

Characterization of a casein kinase which interacts with the rabbit progesterone receptor

Logeat¹,

Cunff²,

Rauch³

et al. 1987

Self Cite

Previous in vivo studies have shown that the rabbit progesterone receptor undergoes two phosphorylation reactions: one basal and a second one which is hormone-dependent. We report here on the presence and characteristics of a kinase activity found in receptor preparations highly purified by immunoaffinity chromatography.1. This kinase activity is not due to the receptor molecule itself since the two proteins may be separated by several chromatographic and immunological methods.2. The presence of the kinase in receptor preparations is not an artefact of the purification procedure. The kinase binds to the receptor as shown by coelution in immunoaffinity experiments and during various chromatographies. This interaction probably takes place in vivo and is not artefactually formed during solubilization of the receptor since the kinase also copurifies with receptors isolated from the uterine nuclei of progestintreated rabbits.3. This enzyme may be classified as a casein kinase since it readily phosphorylates the latter substrate ( K , z 0.15 mg/ml) and is not regulated by cyclic nucleotides, CaZf and calmodulin or phospholipids. Its classification as a casein kinase I or I1 is difficult since on the one hand it is inhibited by heparin, activated by polyamines and may use both ATP and GTP, but on the other hand it modifies only serine residues, and is not inhibited by heparin when the receptor itself is employed as a substrate.4. The kinase which copurifies with the receptor does not mimic in vitro the effects of the hormone-dependent phosphorylation of the receptor observed in vivo: there is no enhancement of kinase activity by the hormone, and the phosphorylated receptor does not exhibit the characteristic "upshift" in its electrophoretic mobility. Thus either this kinase is not the enzyme responsible for the hormone-dependent receptor phosphorylation or, during purification, a factor has been lost which is necessary for retaining the hormone dependency of the reaction.Regulation of various protein kinase activities is one of the major steps in the mechanism of action of most hormones and growth factors acting at the level of the cell membrane (reviews in [I, 21). In the case of the steroid hormones, which act in the cell nucleus, different experimental approaches have implicated protein kinases [3 -161 but no clear scheme of their role has emerged to date.We have studied the phosphorylation of the rabbit progesterone receptor in vivo [I 71. Two phosphorylation reactions were observed: one basal, in the absence of hormone, and a second one which was hormone-dependent and characterized by a slight change in the electrophoretic mobility of receptor in sodium dodecyl sulfate/polyacrylamide gels. As shown in the present paper, a kinase activity is detected in preparations of highly purified receptor and this observation led us to ask the following questions: (a) Is this kinase activity due to the receptor molecule itself and, if not, is the copurification of the kinase with the receptor an artefact or is it related to a ...