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O Wrange

Publications and source records attributed to O Wrange.

At least 73 records · Page 4Linked to original sources
PubMed1979

Purification of the glucocorticoid receptor from rat liver cytosol.

The [3H]-triamcinolone acetonide-labeled glucocorticoid receptor from rat liver cytosol was purified to 85% homogeneity according to sodium dodecyl sulfate gel electrophoresis. It consisted of one subunit with a molecular weight of 89,000 and had one ligand-binding site per molecule. The purification involved sequential chromatography on phosphocellulose, DNA-cellulose twice, and Sephadex G-200. Between the two chromatography steps on DNA-cellulose, the receptor was heat activated. The receptor was affinity eluted from the second DNA-cellulose column with pyrodixal 5'-phosphate. The purification achieved in the first three chromatographic steps varied between 60 and 95% homogeneity in different experiments. After chromatography on the second DNA-cellulose column, the steroid.receptor complex had a Stokes radius of 6.0 nm and a sedimentation coefficient of 3.4 S in 0.15 M KCl. In the absence of KCl, the sedimentation coefficient was 3.6 S. After concentration on hydroxylapatite, the steroid.receptor complex was analyzed by isoelectric focusing in polyacrylamide gel. The radioactivity was shown to focus together with the major protein band with pI 5.8. Following limited proteolysis with trypsin, the radioactivity, together with the major protein band, focused at pI 6.2 as previously described for the unpurified steroid.receptor complex.

Animals↗
PubMed1979

A comparison of the glucocorticoid receptor in cytosol from rat liver and hippocampus.

The [3H]corticosterone- and [3H]dexamethasone-binding proteins in cytosol from liver and hippocampus of the rat were compared by isoelectric focusing analysis in slabs of polyacrylamide gel. A single peak of radioactivity with a pI of 6.1--6.2 was obtained during analysis of cytosol from both liver and hippocampus using either corticosterone or dexamethasone as radiolabelled ligand, provided the tissue was carefully perfused with buffer prior to preparation of cytosol. Rat serum or insufficiently perfused tissue contained a corticosterone-binding component with pI of 5.2--5.5 representing corticosteroid-binding globulin. Limited trypsin digestion resulted in fragmentation of the dexamethasone- and corticosterone-binding protein in cytosol from liver and hippocampus. Incubation of radiolabelled cytosol with 0.5 microgram of trypsin/A280--310nm of cytosol gave a sharp radioactive peak with a pI of 5.9--6.1 when analyzed by isoelectric focusing; when 5.0 microgram of trypsin/A280--310nm of cytosol was used, a double peak with pI values of 5.9--6.1 and 6.3--6.5, respectively, was seen. The same trypsin-induced peaks were seen with both [3H]dexamethasone and [3H]corticosterone as ligands. The substrate specificity and the sensitivity of this glucocorticoid binder for limited trypsin digestion is in good agreement with what was previously found for the glucocorticoid receptor in rat liver cytosol. It is concluded that cytosol from liver and hippocampus contains an identical or very similar receptor for glucocorticoid hormones.

Animals↗
PubMed1978

Estradiol receptor analysis in human breast cancer tissue by isoelectric focusing in polyacrylamide gel.

Isoelectric focusing in polyacrylamide gel combined with limited proteolysis is a simple and specific method for quantitation of estradiol receptors in breast cancer tissue. At least eight different samples can be analyzed simultaneously on one gel, and the whole procedure, including sample preparation, takes less than 7 hr. In comparison with sucrose gradient centrifugation, isoelectric focusing is more sensitive, possibly due to the short time (1.5 to 2 hr) needed for the analysis. Furthermore, only one incubation with tritium-labeled estradiol is needed for an analysis, which means that a smaller amount of tumor tissue is needed than for most other methods. This fact allows analysis of the estrogen receptor content in tumor material obtained from fine-needle biopsy.

Breast Neoplasms↗
PubMed1977

Formation and characteristics of hepatic dexamethasone-receptor complexes of different molecular weight.

The dexamethasone-binding receptor protein in rat liver cytosol has a Stokes radius of 61 A and a sedimentation coefficient of 4.0 S. In contrast, cell nuclei labelled with [3H]dexamethasone in vivo or in vitro (reconstitution experiments with [3H]dexamethasone-labelled cytosol and isolated unlabelled nuclei) contain a high-salt-extractable dexamethasone-receptor complex with a Stokes radius of 30-36 A and a sedimentation coefficient of 3.2 S. Exposure of liver homogenate or 1000 X g homogenate supernatant to low ionic strength during preparation of cytosol resulted in conversion of the 61 A to a 36 A complex very similar to the intranuclear form of dexamethasone receptor. 61 leads to 36 A complex-converting activity was present in both the 100 X g-10 000 X g sediment of liver homogenate, from which it could be extracted by hypotonic media, and in the liver cell nuclei, from which it could be extracted by hypertonic media. Mild digestion of the 61 A dexamethasone-receptor complex with trypsin also gave rise to a complex with a Stokes radius of 36 A. Reconstitution experiments with isolated liver cell nuclei indicated that both the 61 A and 36 A dexamethasone-receptor complexes were taken up by the nuclei; reextraction of the nuclei incubated with the 61 A complex revealed that this form had been converted to the 30-36 A complex. Further digestion of the 61 and 36 A [3H]dexamethasone-receptor complexes with hypotonic extract of the 1000 X g-10 000 X g sediment of liver homogenate or with trypsin resulted in formation of a third complex with Stokes radius of 19 A and a sedimentation coefficient of 2.5 S. The approximate molecular weights of the 61, 36 and 19 A dexamethasone-receptor complexes were calculated as 102 000, 46 000 and 19 000, respectively, and the frictional ratios of the molecules as 1.84, 1.38 and 1.00, respectively. It is concluded that the nuclear 30-36 A dexamethasone-receptor complex is formed from the cytosol 61 A complex by proteolytic digestion and that this latter protein contains at least two sites with a relatively high sensitivity to protelytic cleavage.

Animals↗
PubMed1977

Interactions of corticosterone, 5alpha-dihydrocorticosterone and dexamethasone with proteins in rat-liver cytosol.

The intracellular binding of [3H]corticosterone and [3H]dexamethasone and their metabolites to macromolecules in rat liver cytosol was studied in vivo and in vitro. The macromolecules binding corticosterone and its metabolites were characterized as (a) a steroid conjugate-binding (Stokes radius 2.5 nm and sedimentation coefficient 4.1 S in high ionic strength; pI 8.7, (b) transcortin and (c) a glucocorticoid "receptor". Competition experiments indicate that corticosterone and dexamethasone bind to the same site of the glucocorticoid receptor molecule. Different Stokes radii between the corticosterone-receptor and the dexamethasone-receptor complexes (6.9 and 6.3 nm, respectively, in high ionic strength) indicate that the two ligands induce different conformations of the receptor protein. This may be of importance when explaining the qualitative differences between the cellular effects of natural and synthetic glucocorticoids. 5alpha-Dihydrocorticosterone, on the other hand, competed to a very limited extent with dexamethasone for binding sites on the receptor. An assay of the inductive effect on liver tyrosine aminotransferase and tryptophan oxygenase indicated that 5alpha-dihydrocorticosterone was practically devoid of glucocorticoid activity. It is concluded that 5alpha-dihydrocorticosterone probably does not act as the mediator of corticosterone action in rat liver.

Adrenalectomy↗
PubMed1976

Characteristics of the corticosterone-receptor complex in rat liver cytosol.

Using a gel filtration on Sephadex G-150 in low ionic strength, it was possible to separate a corticosterone-binding protein in rat liver cytosol from corticosteroid-binding globulin after incubation of cytosol with [3H]corticosterone. The corticosterone-protein complex ("alpha-Complex") had a sedimentation coefficient of 8-9 S in low ionic strength. In high ionic strength, the alpha-Complex rapidly dissociated with a half-life of 15 h, compared to a half-life of 31 h for the hepatic dexamethasone-receptor complex under identical conditions (0 degrees C). The alpha-Compelx was saturable with an excess of unlabelled corticosterone of dexamethasone and was sensitive to heat and protease digestion. It is stressed that quantitation of the corticosterone-receptor complex must include separation of the receptor from corticosteroid-binding globulin as this protein binds corticosterone with high affinity and with a saturable amount of binding sites.

Animals↗