共查询到20条相似文献,搜索用时 8 毫秒
1.
Mohammed Kalimi John Hubbard 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,719(3):488-494
0.1–0.3 M sodium thiocyanate greatly enhanced the rate of inactivation of unbound rat hepatic glucocorticoid receptors in vitro at 4°C. Prior treatment of the unbound glucocorticoid receptor with 10 nM molybdate (at 25°C for 30 min) protected the receptor from 0.3 M KCl, but not from 0.3 M NaSCN inactivation. When the [3H]dexamethasone-receptor complex was examined on sucrose density gradients containing 0.1 M NaSCN, the receptor sedimented as a 4 S complex rather than the 7 S form observed in 0.1 M KCl gradients. NaSCN was found to be more effective in the extraction of both in vivo and in vitro nuclear-bound [3H]dexamethasone-receptor complexes than KCl. At a concentration of 0.3 M, NaSCN extracted most of the specific nuclear-bound receptor. 50 mM NaSCN significantly blocked the thermal activation of preformed [3H]dexamethasone-receptor complexes. The chaotropic salt, NaSCN, appears therefore to have significant effects on glucocorticoid receptors in vitro. In addition, NaSCN appears to be a useful agent in quantitative extraction of steroid from nuclear-bound steroid-receptor complexes. 相似文献
2.
Activation of the glucocorticoid-receptor complex 总被引:2,自引:0,他引:2
A crucial step in the interaction of glucocorticoids with target cells is the activation step, which involves a conformational change in the cytoplasmic glucocorticoid-receptor protein complexes and facilitates their binding to the cell nucleus. Activation can be quantified by measuring the ability of glucocorticoid-receptor complexes to bind to polyanions, such as DNA-cellulose, and unactivated complexes can be separated from activated complexes by rapid ion exchange chromatography using diethylaminoethyl (DEAE)-Sephadex or DEAE-cellulose. Activation occurs in vivo under physiological conditions and the rate of activation of cytoplasmic glucocorticoid-receptor complexes can be enhanced in vitro by physical manipulations (elevated temperature, increased ionic strength, dilution). In vitro studies suggest that activation is a regulated process and a low molecular weight component termed modulator, which has been identified in rat hepatic cytosol, inhibits activation. Additional studies employing phosphatase inhibitors, such as molybdate, and purified calf intestinal alkaline phosphatase suggest that either the receptor protein or a regulatory component is dephosphorylated during activation. Results obtained with specific chemical probes suggest that activation results in the exposure of basic amino acid residues consisting minimally of lysine, arginine, and histidine. Pyridoxal 5'-phosphate, a specific probe for lysine residues, exerts dual effects on glucocorticoid-receptor complexes, since it stimulates the rate of activation and also inhibits the binding of previously activated complexes to nuclei or DNA-cellulose. The ability of 1,10-phenanthroline, a metal chelator, to inhibit the DNA-cellulose binding of activated complexes suggests that a metal ion(s) located at or near the DNA binding site may become exposed as a consequence of activation. Collectively, the results of these various experiments suggest that activation is a regulated biochemical phenomenon with physiological significance. 相似文献
3.
Gian Paolo Rossini 《Molecular and cellular biochemistry》1985,68(1):67-78
Summary The investigation on hydrodynamic parameters of molybdate-stabilized glucocorticoid-receptor complexes from HeLa cell cytosol permitted resolution of four distinct forms. The first one could be detected in concentrated cytosols at low salt concentrations, and had the following properties: sedimentation coefficient = 9 S; R
s = 9.3 nm; M
r = 357,800; f/f
o = 1.83; axial ratio (prolate ellipsoid) = 16. When these cytosol extracts were diluted, a second form could be detected with sedimentation coefficient = 8.3 S; R
s = 9.05 nm; M
r = 320,700;f/f
o = 1.84; axial ratio = 16. Under high salt conditions, glucocorticoid-receptor complexes in concentrated cytosol had the following properties: sedimentation coefficient = 6.4 S; R
s, = 6.7 nm; M
r = 183,100;f/f
o = 1.64; axial ratio = 12. When either these cytosol extracts were diluted, or glucocorticoid-receptor complexes were subjected to repeated analysis, a fourth form was detected with sedimentation coefficient = 3.76 S; R
s = 5.67; M
r = 91,000; f/f
o = 1.75; axial ratio = 14. Besides salt concentration and dilution, the time elapsed between sample dilution and analysis appeared to affect the hydrodynamic properties of glucocorticoid-receptor complexes. On the basis of our findings, it has been concluded that the most likely structure of molybdate-stabilized glucocorticoid-receptor complexes of HeLa cell cytosol can be represented by association of monomers in homodimers, and homotetramers. A homotrimer form could not be deduced from our findings, and the 320,700 glucocorticoid-receptor complex we observed has been suggested to represent an unresolved mixture of trimers and tetramers. 相似文献
4.
Simonetta Sperti Lucio Montanaro Fioretta Rambelli Mariacristina Zamboni 《Bioscience reports》1986,6(10):901-908
Alpha-sarcin and gelonin, two proteins which inactivate the 60S ribosomal subunit, interact with Cibacron blue and bind to blue dextran-Sepharose, from which they are partially desorbed by nucleoside triphosphates and, more efficiently, by homopolynucleotides. It is further shown that the two proteins bind to poly(U)-Sepharose and that homopolynucleotides protect dilute solutions of both inhibitors from inactivation. It is suggested that the presence of a polynucleotide site on alphasarcin is related to its ribonuclease activity. The existence of a similar site on gelonin might be a clue to its yet unknown mechanism of action. 相似文献
5.
Evidence that pH induced activation of the rat hepatic glucocorticoid-receptor complex is irreversible 总被引:2,自引:0,他引:2
The possible reversibility of pH induced activation of the glucocorticoid-receptor complex was studied. Generally, this was accomplished by activating rat liver cytosol at pH 8.5 (15 degrees C, 30 min), and then returning it to pH 6.5 for a second incubation (15 degrees C, 30 min). Activation was quantitated by measuring the binding of [3H]triamcinolone acetonide [( 3H]TA)-receptor complexes to DNA-cellulose. When cytosol was incubated at pH 6.5, only 4.1% of the [3H]TA-receptor complexes bound to DNA-cellulose. However, 39.2% of the complexes bound when the cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 47.0% of the steroid-receptor complexes bound. Thus, according to the DNA-cellulose binding assay, pH induced activation was irreversible. In order to visualize both activated and unactivated [3H]TA-receptor complexes during this process, diethylaminoethyl (DEAE)-cellulose chromatography was performed. When cytosol was incubated at pH 6.5, only 19.6% of the [3H]TA-receptor complexes were eluted in the activated form from DEAE-cellulose. However, 67.5% of the complexes were eluted in the activated form when cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 74.9% of the steroid-receptor complexes were eluted in the activated form. Thus, DEAE-cellulose chromatography also showed that pH induced activation was irreversible. This is the first known report that the combination of DNA-cellulose binding and DEAE-cellulose chromatography have been used to study pH induced activation of the glucocorticoid-receptor complex. By these criteria, we conclude that in vitro pH induced activation is irreversible. 相似文献
6.
雄激素受体片段(氨基酸:359 ̄732)以及糖皮质激素受体片段(氨基酸:396 ̄548)以与GST融合形式在大肠杆菌中分别表达为GST-AR和GST-GR两种融合蛋白,它们含有DNA结合结构域和一些旁侧氨基酸,凝胶阻滞分析表明能与雄激素/糖皮质激素应答元件(ARE/GRE)在体外结合。本文报道在大鼠前列腺中发现有抑制GST-AR以及GST-GR与ARE/GRE结合的因子,抑制作用与ARE/GRE来 相似文献
7.
Fioretta Rambelli Maurizio Brigotti Simonetta Sperti Lucio Montanaro 《Bioscience reports》1987,7(9):737-743
Diphtheria toxin fragment A interacts with Cibacron blue in solution, although it is not retained by blue Sepharose columns. Difference spectral titration of fragment A with the dye gives a dissociation constant of the order of 10–5 M and a 11 stoichiometry for the complex. In equilibrium dialysis experiments Cibacron blue behaves as a competitive inhibitor of the binding of NAD to diphtheria toxin fragment A. The dye inhibits in a non-competitive way the fragment A-catalysed transfer of ADP-ribose from NAD to elongation factor 2 (EF2). By affinity chromatography on blue Sepharose a binding of EF2 and of ADP-ribosyl-EF2 with the dye is also demonstrated. GDP, GTP and GDP(CH2)P are able to displace EF2 from blue Sepharose. 相似文献
8.
Cibacron blue is a potent inhibitor of 3-HBA-6-hydroxylase at a concentration < 1 μM. Kinetic analyses revealed that at a concentration below 0.5 μM the dye behaves as an uncompetitive inhibitor with respect to 3-HBA and competes with NADH for the same site on the enzyme. The alteration of the near-UV CD spectrum and quenching of the emission fluorescence of the enzyme by cibacron blue indicates a significant alteration in the environment of aromatic amino acid residues due to a stacking interaction and subtle conformatiodnal changes in the enzyme. The concentration-dependent quenching of the intrinsic fluorescence of the enzyme by cibacron blue was employed to determine the binding parameters such as association constant (Ka) and stoichiometry (r) for the enzyme-dye complex. 相似文献
9.
The glucocorticoid receptor-glucocorticoid complex of the hepatic cytosol need undergo an "activation" to enable its binding to nuclei, chromatin, or stripped DNA. The conditions of this activation have been studied using native calf thymus DNA absorbed to cellulose. At low ionic strength, activation is very slow at 0 degrees, but, takes place rapidly at 25 degrees, reaching completion at 1 hour. Addition of 10 mm CaCl2 or 150 mm NaCl increases the rate of activation of the receptor at 0 degrees. Neither magnesium nor manganese ions can replace calcium with respect to enabling activation of the steroid-receptor complex to occur at low temperatures. Isofocusing studies reveal that the major component of the unactivated steroid-receptor complex has an isoelectric point of 7.1. Incubation of the steroid-receptor complex at 25 degrees for 30 min leads to its conversion to a form with an isoelectric point of 6.1 concurrent with the development of its ability to bind to DNA-cellulose. Sucrose density gradient analysis reveals that no detectable alteration in the sedimentation coefficient of the steroid-receptor complex occurs during its activation. MnCl2 (20mm) effeciently precipitates the unactivated hormone-receptor complex and to a lesser degree, precipitates the activated hormone-receptor complex. 相似文献
10.
Mohammed Kalimi 《Analytical biochemistry》1978,86(1):166-174
Protamine sulfate was found to precipitate completely the nonactivated [3H]-dexamethasone-receptor complex of rat liver. This observation was then used as the basis of a method to separate activated from nonactivated complex. Thus, addition of 10 mg/ml of protamine sulfate to the rat hepatic cytosol [3H]dexamethasone-receptor complex, incubated at 0–4°C for 2 hr, resulted in the complete precipitation of [3H]dexamethasone-receptor complex. The remaining supernatant obtained on centrifugation at 800g was unable to bind either to nuclei or to DNA-cellulose. An increase in temperature to 25°C or the addition of 10 mm CaCl2 to the cytosol resulted in the appearance of activated [3H]dexamethasone-receptor complex in the supernatant obtained by addition of protamine sulfate. This was determined by characteristic binding to nuclei or DNA cellulose and by pI. Protamine sulfate could not affect the separation of activated [3H]dexamethasone-receptor complex at salt concentrations above 100 mm NaCl. This procedure therefore had to be carried out under conditions of relatively low ionic strength. Finally, a one-step rapid method is described for the separation of activated [3H]dexamethasone-receptor complex from nonactivated receptor complex. The homogeneous population of activated complex thus obtained should have considerable applicability in studies of the mechanisms of in vitro glucocorticoid-receptor activation. 相似文献
11.
Interaction of Prussian blue nanoparticles with bovine serum albumin: a multi-spectroscopic approach
Hongyu Zhou Xin Shi Yuanjie Fan Zhiying He Ling Ye 《Journal of biomolecular structure & dynamics》2018,36(1):254-261
Owning to their exceptional properties, Prussian blue nanoparticles (PBNPs) are promising in a variety of biomedical applications. In this scenario, understanding of how PBNPs interact and behave in biological systems is essential. Herein, the interaction of PBNPs with protein was investigated. Specifically, the citric acid stabilized PBNPs with a size of 10 nm were synthesized and characterized. The interactions of these PBNPs with the model protein, bovine serum albumin (BSA), were then probed by spectroscopic methods. It was found that the BSA intrinsic fluorescence was quenched upon addition of PBNPs due to the static interaction, suggesting the binding of PBNPs with BSA. Moreover, the synchronous fluorescence and circular dichroism spectra indicated the conformational change of BSA due to the presence of PBNPs. 相似文献
12.
Salt-induced transformation of the glucocorticoid-receptor complex in the presence of molybdate 总被引:1,自引:0,他引:1
The glucocorticoid hormone-receptor complex has been shown to exist in several forms. The transformation status of various forms of the complex isolated from rat thymus cytosol in the presence of molybdate was determined. The non-transformed receptor had a higher affinity for DEAE-cellulose than the transformed receptor. The rate at which the non-transformed complex was transformed to a smaller form with a low affinity for DEAE-cellulose by exposure to salt was greater in the absence of molybdate than in its presence. We conclude that salt-induced transformation of the complex is retarded but not prevented by molybdate and is associated with subunit dissociation. 相似文献
13.
Hydrodynamic and biochemical correlates of the activation of the glucocorticoid-receptor complex 总被引:2,自引:0,他引:2
Possible changes in the size and shape of the glucocorticoid-receptor complex (GRC) following activation remain poorly documented, due to the lability and possible activation of the receptor during the determination of these hydrodynamic parameters. In the present study molybdate was used to stabilize the GRC, thus preventing these uncontrolled transformations. Cytosol prepared from mouse whole brains was incubated for 18 h at 0-2 degrees C with [3H]triamcinolone acetonide (+/- molybdate). Activation was then initiated by incubation at 22 degrees C for variable times and quenched at 0 degree C by adding molybdate. The Stokes radius and sedimentation coefficient of the GRC declined from 77 A and 9.2 S before activation to 58 A and 3.8 S after activation. These measurements remained consistent after recycling GRC between sedimentation and gel filtration procedures and correspond to a 3-fold reduction in the relative molecular mass. The loss and formation of the 297 and 92 kDa species, respectively, after different durations of activation correlated nearly perfectly with increased binding of GRC to DNA-cellulose (DNA-C). The observed size change also correlated well with decreased adsorption to DEAE-cellulose filters (DE-81) and increased adsorption to glass fiber filters (GF/C). The increased adsorption to GF/C may reflect an increase in hydrophobicity which, with extended durations of activation, leads to increased aggregation and reduced binding to DNA-C, but not to a change in adsorption to DE-81. We propose that during activation the 297 kDa form of the GRC splits to form a 92 kDa species that displays an increased affinity for DNA. 相似文献
14.
15.
目的:探讨热休克蛋白90(HSP90)在糖皮质激素(GC)信号通路中的作用及其对气道黏蛋白(MUC)5AC表达的调控机制。方法:体外培养气道上皮细胞株BEAS-2B,给予HSP90特异性阻断剂格尔德霉素(GA)和地塞米松(DEX)刺激,比较各组MUC5AC的含量,糖皮质激素受体(GR)的核蛋白表达和GR结合活性。结果:与对照组相比,DEX组的MUC5AC mRNA和蛋白水平降低,伴GR核蛋白水平增高(P0.05);与DEX组相比,GA+DEX组MUC5AC mRNA和蛋白水平升高,GR核蛋白水平降低。放射配体结合实验显示,与对照组相比,GA组的受体最大结合容量(Bmax)降低,平衡解离常数(Kd)升高。结论:HSP90可参与GC及GR的抗气道黏液高分泌效应,其抑制剂可阻断上述效应,该效应是通过降低GR的结合活性来调控的。 相似文献
16.
R. A. Karnachuk M. A. Bol’shakova M. V. Efimova I. F. Golovatskaya 《Russian Journal of Plant Physiology》2008,55(5):597-602
The effects of blue light (BL) and jasmonic acid (JA) on morphogenesis of Arabidopsis thaliana (L.) Heynh seedlings of genotypes Col and Ler and their mutants, namely, axr1-3 and jar1-1 mutants resistant to IAA and JA, respectively, and a CRY1 photoreceptor-deficient mutant hy4 were studied. Both 1 μM JA and BL exposure retarded hypocotyl growth of Ler, Col, and jar1-1 seedlings, whereas JA had no effect on hypocotyl growth of axr1-3, but the suppression of hypocotyl growth of this mutant by BL was even more noticeable than that of Ler, Col, and jar1-1. JA and BL applied simultaneously inhibited hypocotyl growth of axr1-3 and especially of Ler, Col, and jar1-1 more than either of factors applied separately. The hy4 mutant did not respond to BL, whereas JA stimulated its hypocotyl growth. JA did not change the cotyledon size of Col, axr1-3, and jar1-1 and reduced the cotyledon size of Ler and hy4. BL enhanced the cotyledon growth of all wild-type and mutant plants used in the study. The cotyledon sizes of all plants except Ler were also increased when JA and BL were applied together. Some of the growth responses correlated with the endogenous IAA and ABA contents. Thus, for example, the hypocotyl and cotyledon growth retardation of Ler seedlings in the presence of JA correlated with a reduced level of free IAA and a considerable increase in the free ABA level in plants grown both in darkness and in BL. Under other growth conditions, no correlation between the endogenous IAA and ABA levels and A. thaliana seedling growth was noted. The interaction between the signal transduction pathways triggered by BL and JA at the early stages of arabidopsis morphogenesis is discussed on the basis of Col, Ler, axr1-3, and jar1-1 hypocotyl growth responses. 相似文献
17.
G P Rossini 《Biochimica et biophysica acta》1987,924(1):119-126
The contention that transformation of steroid-receptor complexes is represented by dissociation of receptor oligomers was tested by comparing sedimentation and DNA binding properties of glucocorticoid-receptor complexes from HeLa cell cytosol under several conditions. Transformation of glucocorticoid-receptor complexes could be induced by heat, and/or salt treatment of cytosolic extracts, but not by dilution. Heat-induced transformation of receptor complexes was also confirmed by DEAE-cellulose chromatography. Analysis of cytosolic extracts showed that sedimentation and DNA binding properties of glucocorticoid-receptor complexes did not correlate. Both oligomeric and monomeric receptor complexes, in fact, were found to be either transformed, or untransformed, depending on the treatments cytosolic extracts underwent, before being subjected to analysis. We then concluded that release of glucocorticoid receptor monomers cannot account for their transformation to a DNA-binding form in vitro, and suggested that exposure of positive charges on the surface of receptors in the course of transformation occurs in some region of the glucocorticoid receptor which is not involved in interactions between the proteinaceous components of oligomers. 相似文献
18.
糖皮质激素非基因组效应及其信号转导机制 总被引:2,自引:0,他引:2
糖皮质激素具有多种重要的生理和药理作用,其经典作用途径为“基因组机制”,通过调节基因转录发挥作用.近年来,其“非基因组机制”在生理和药理学方面的作用越来越受重视.在这一作用途径中,可能有多种受体、激酶、信号分子的参与,“基因组机制”和“非基因组机制”间还可能存在交互调节,对非基因组机制进行深入研究有利于糖皮质激素的临床合理应用. 相似文献
19.