scholarly journals Physical measurements of the liver glucocorticoid receptor

1978 ◽  
Vol 169 (2) ◽  
pp. 445-448 ◽  
Author(s):  
G Litwack ◽  
M H Cake ◽  
R Filler ◽  
K Taylor
Keyword(s):  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Axial Ratio ◽  
Steroid Receptor ◽  
Sucrose Density Gradient Centrifugation ◽  
Uniform Size ◽  
Receptor Complexes ◽  
Physical Measurements

Physical measurements were made on the cytosolic form of the liver [3H]dexamethasone receptor. These include a Stokes radius of 3.5 nm, determined by gel filtration, and sedimentation coefficients of 5.1 and 7-8S, by sucrose-density-gradient centrifugation. From these measurements, the following physical properties were calculated: apparent mol. wt. 78000 (the 5.1 S form); D app. 6.1 × 10(-7) cm2-S-1; f/fo 1.25; axial ratio 4.7; these indicate a globular protein. Measurements of sedimentation coefficient of cytosol steroid-receptor complexes previously subjected to various activating conditions gave different values and lead to the conclusion that the mechanism of activation in vitro enabling the steroid-receptor complex to bind to DNA is more complex than simple disaggregation to a uniform size.

Notizen: Physical Parameters and Possible Regulation of ζ-Aminolevulinic Acid Synthetase

1978 ◽  
Vol 33 (9-10) ◽  
pp. 796-798 ◽  
Author(s):  
Dhirendra L. Nandi
Keyword(s):  
Aminolevulinic Acid ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Axial Ratio ◽  
Enzymic Activity ◽  
Partial Specific Volume ◽  
Physical Parameters ◽  
Sucrose Density Gradient Centrifugation ◽  
Physical Measurements

Abstract Physical measurements were made on the ζ-aminolevulinic acid synthetase from Rhodopseudomonassph eroides. These include a Stokes radius of 3.8 nm, determined by gel filtration, and sedimentation coefficient of 5.46 S by sucrose density gradient centrifugation. From these measurements and the value of partial specific volume of 0.732 ml/g deter­mined from the amino acid composition, the following physical constants were calculated: molecular weight, 88000; diffusion coefficient, 5.65 × 10-7 cm2 s-1 ; frictional ratio, 1.30; axial ratio, 5.0. The enzyme is inhibited by more than 50% by glyceraldehyde 3-phosphate (1 mᴍ) , pyruvate (0 .02 ᴍ) , α-ketoglutarate (0.02 ᴍ) , urea (0 .8 ᴍ) , CoCl2 (0.2 mᴍ) and hemin (5 µᴍ). The effect of these inhibitors on the possible regulation of this enzymic activity is discussed.

PRELIMINARY CHARACTERIZATION OF AN ANDROGEN-MACROMOLECULAR COMPLEX FROM THE RAT VENTRAL PROSTATE

1969 ◽  
Vol 62 (1) ◽  
pp. 153-164 ◽  
Author(s):  
Olav Unhjem ◽  
Kjell J. Tveter ◽  
Asbjørn Aakvaag
Keyword(s):  
Proteolytic Enzymes ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Ventral Prostate ◽  
Macromolecular Complex ◽  
Sucrose Density Gradient Centrifugation

ABSTRACT Following administration of (1,2-3H)-testosterone to castrated rats or incubation of prostatic tissue with the same steroid, a gel filtration technique has been used for the isolation of a soluble steroid-macromolecular complex from the tissues. Subsequent steroid analyses revealed that 5α-androstan-17β-ol-3-one was the major component associated with the macromolecules both in the in vivo and by in vitro experiments. The complex is destroyed by proteolytic enzymes like trypsin and pronase, but is unaffected by DNase and RNase. The complex is excluded from G-200 as well as P-300 gel beds. By sucrose density gradient centrifugation two macromolecular components were found associated with radioactivity. The largest component had a sedimentation coefficient of 9.3 S and probably corresponds to the macromolecular complex demonstrated by gel filtration, whereas the smaller component had a sedimentation coefficient of 4.5 S and might represent an association of steroids with serum albumin.

Purification and some properties of the glutamate dehydrogenase of Salmonella typhimurium

1973 ◽  
Vol 19 (4) ◽  
pp. 427-438 ◽  
Author(s):  
J. W. Coulton ◽  
M. Kapoor
Keyword(s):  
Salmonella Typhimurium ◽  
Glutamate Dehydrogenase ◽  
Ammonium Sulfate ◽  
Gradient Centrifugation ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient Centrifugation ◽  
Ammonium Sulfate Fractionation ◽  
Sulfate Fractionation

NADP-specific glutamate dehydrogenase (GDH) from Salmonella typhimurium was purified 190-fold by heat treatment, ammonium sulfate fractionation, DEAE-Sephadex chromatography, reverse ammonium sulfate fractionation, and gel filtration. The enzyme proved to be stable to 55 °C, and displayed a pH optimum at 8.6 in the amination reaction. The sedimentation coefficient of GDH, as determined by sucrose density gradient centrifugation, was about 10.3 S. From gel filtration chromatography, the molecular weight and Stokes' radius for the enzyme were estimated at 280 000 daltons and 54 × 10−8 cm, respectively. Unusual resistance was displayed by the enzyme to high concentrations of the protein denaturants, urea, SDS, and guanidine hydrochloride.

Molecular characteristics of cytostatic factors in amphibian egg cytosols

Development ◽  
1989 ◽  
Vol 106 (4) ◽  
pp. 799-808
Author(s):  
E.K. Shibuya ◽  
Y. Masui
Keyword(s):  
Density Gradient ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Molecular Characteristics ◽  
Small Peptides ◽  
Sucrose Density Gradient Centrifugation ◽  
Cytostatic Factor

In amphibians, zygotes microinjected with cytosol of unactivated eggs are arrested at metaphase of mitosis. The factor responsible for this effect has been designated ‘cytostatic factor, (CSF)’. CSF is inactivated by Ca2+ addition to cytosols. During storage of the Ca(2+)-containing cytosols, a stable CSF activity develops. Therefore, the first Ca(2+)-sensitive CSF and the second Ca(2+)-insensitive CSF have been referred to as primary CSF (CSF-1) and secondary CSF (CSF-2), respectively. We have partially purified CSF-1, which had been stabilized with NaF and ATP, and CSF-2 from cytosols of Rana pipiens eggs by ammonium sulphate (AmS) precipitation and sucrose density gradient centrifugation or gel filtration, and investigated their molecular characteristics. CSF-1 was sensitive to protease, but resistant to RNAse, and inactivated within 2 h at 25 degrees C. CSF-1 could be sedimented in a sucrose density gradient from a fresh cytosol or its crude fraction precipitated at 20–30% saturation of AmS, showing the sedimentation coefficient 3S. When analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), all the proteins in partially purified CSF-1 samples entered the gel and were separated into numerous peptide bands. In contrast, CSF-2 was an extremely large molecule, being eluted from Sepharose columns as molecules larger than 2 × 10(6), and failed to enter the gel when analyzed by SDS-PAGE. It could be purified 40 times from cytosols. CSF-2 was a highly stable molecule, being neither inactivated nor dissociated at pH 11.5 or by 4M-NaCl and LiCl and 8 M-urea. It was also resistant to RNAse treatment. However, CSF-2 could be broken down into small peptides of variable sizes by trypsin, alpha-chymotrypsin, and papain, but not by S. aureus V8 protease, although it was less sensitive to proteases than CSF-1. The dose-dependency test showed that the activity of CSF-2 is independent of its concentration and that an amount of CSF-2 could cause cleavage arrest earlier when injected into a blastomere in a larger volume.

scholarly journals Deoxyribonucleic acid poymerase of BHK-21/C13 cells. Heterogeneity, molecular asymmetry and subcellular distribution of the enzymes

1975 ◽  
Vol 145 (2) ◽  
pp. 225-232 ◽  
Author(s):  
R K Craig ◽  
H M Keir
Keyword(s):  
Dna Polymerase ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Dna Polymerase I ◽  
Sucrose Density Gradient Centrifugation ◽  
Molecular Weights ◽  
Molecular Asymmetry ◽  
Polymerase I ◽  
Dna Polymerase Ii

Nuclear and cytoplasmic fractions were prepared from exponentially-growing BHK-21/C13 cells; DNA polymerase was extracted from them and analysed by gel filtration and sucrose-density-gradient centrifugation. DNA polymerase I is heterogeneous comprising species covering a considerable range of molecular weights. These have been tentatively identified as four subspecies of apparent molecular weights 900000-1000000 (IA), 460000-560000 (IB), 270000-320000 (IC) and 140000-200000 (ID), as assessed by gel filtration through Sepharose 6B. DNA polymerase II has a mol.wt. of 46000 +/- 4000 as assessed by gel filtration on Sepharose 6B, and 48000 +/- 2000 as assessed by gel filtration on Sephadex G-100. Sedimentation analyses on sucrose density gradients showed that the DNA polymerase I species had sedimentation coefficients predominantly in the range 6-8 S. DNA polymerase II had predominantly a sedimentation coefficient of 3.2 S although a component with lower sedimentation coefficient was found. The lack of correlation between the molecular weights derived from gel filtration and the sedimentation coefficients is attributed to molecular asymmetry. DNA polymerase I was found to be associated predominantly with the cytoplasm although certain types of nuclear preparation contained large amounts of it. DNA polymerase II was found to be mostly if not exclusively in nuclear preparations.

scholarly journals Chemical cross-linking of chick oviduct progesterone-receptor subunits by using a reversible bifunctional cross-linking agent

1979 ◽  
Vol 181 (1) ◽  
pp. 201-213 ◽  
Author(s):  
M E Birnbaumer ◽  
W T Schrader ◽  
B W O'Malley
Keyword(s):  
Progesterone Receptor ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Cross Linking ◽  
Sucrose Density Gradient Centrifugation ◽  
Receptor Proteins ◽  
The Cross

Chick oviduct progesterone-receptor proteins were treated in cytosol with the reversible cross-linking reagent methyl 4-mercaptobutyrimidate. The product of the reaction was a 7S complex that could be detected and recovered after sucrose-density-gradient centrifugation in 0.3M-KCl. The extent of the reaction was dependent on the concentration of methyl 4-mercaptobutyrimidate and independent of the presence of bound hormone, since unlabelled receptors could also be cross-linked. The cross-linking reaction required conditions in which the cytosol 6S complex was preserved. A Stokes radius of 7.3 nm was determined by gel filtration in Agarose A-1.5 m in 0.3 M-KCl. The sedimentation coefficient, which was also determined in 0.3 M-KCl, allowed us to calculate a mol. wt. of 228,000. We were also able to cross-link partially purified receptor forms isolated by using an Agarose A-15 m column. On reduction with beta-mercaptoethanol the complex broke down to 4S monomers that were identified by DEAE-cellulose and phosphocellulose chromatography, adsorption on DNA-cellulose and gel filtration in an Agarose A-1.5 m column. In most cases, A and B receptor proteins were released in equivalent amounts, implying that the cross-linked form was an A-B complex.

scholarly journals Uptake of oestradiol by the rabbit hypothalamus. Specificity of binding by nuclei in vitro

1970 ◽  
Vol 118 (1) ◽  
pp. 93-97 ◽  
Author(s):  
Gerald J. Chader ◽  
Claude A. Villee
Keyword(s):  
Specific Binding ◽  
Gradient Centrifugation ◽  
Significant Proportion ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Cerebellar Nuclei ◽  
Supernatant Fraction ◽  
Sucrose Density Gradient Centrifugation ◽  
Hypothalamic Nuclei

The binding of [6,7-3H]oestradiol-17β to subcellular fractions of the hypothalamus and the cerebellum of the rabbit was studied in vitro. Uptake of steroid was higher in hypothalamic nuclei than in cerebellar nuclei. Lower binding was observed in other fractions of both tissues. After dialysis of the fractions, hypothalamic nuclei retained a high percentage of oestradiol whereas cerebellar nuclei lost most of the bound steroid. Supernatant fractions of both tissues retained a significant proportion of label after dialysis and after gel filtration on Sephadex G-200. No specific binding was observed in these fractions when subjected to sucrose-density-gradient centrifugation. Purification of nuclei followed by incubation with labelled oestradiol in the absence of the supernatant fraction resulted in loss of binding of steroid by hypothalamic nuclei. Incubation of the purified hypothalamic nuclei with supernatant fraction maintained the binding specificity of hormone retention.

scholarly journals Hydrodynamic and pharmacological characterization of putative α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate-sensitive l-glutamate receptors solubilized from pig brain

1994 ◽  
Vol 300 (2) ◽  
pp. 365-371 ◽  
Author(s):  
T Y Wu ◽  
Y C Chang
Keyword(s):  
Binding Sites ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Glutamate Binding ◽  
Stokes Radius ◽  
Triton X

L-[3H]Glutamate binding sites with characteristics resembling that of membrane-bound alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-subtype L-glutamate receptors have been solubilized from pig brain synaptic junctions by Triton X-114. Binding of [3H]AMPA to these soluble sites in the presence of KSCN results in a curvilinear Scatchard plot that can be resolved into a high-affinity component and a low-affinity component. These Triton-X-114-solubilized sites can be further separated into two species of binding sites by gel-filtration chromatography or sucrose-density-gradient centrifugation. The pharmacological profiles of these two species of binding site are almost identical, and the rank orders of potency for glutamatergic drugs in displacing L-[3H]glutamate binding to these sites are quisqualate > 6,7-dinitroquinoxaline-2,3-dione > 6-cyano-7-nitroquinoxaline-2,3-dione > AMPA > L-glutamate > kainate >> N-methyl-D-aspartate = L-2-amino-4-phosphonobutyrate. Both sites are found to bind [3H]AMPA, and in the presence of KSCN the binding activities are significantly enhanced. Analysis of the hydrodynamic behaviour of these binding sites by sucrose-density-gradient centrifugation in H2O- and 2H2O-based solvents and gel-filtration chromatography has revealed that one of these sites (Stokes radius 8.3 nm, sedimentation coefficient 18.5 S) consists of 562 kDa protein and 281 kDa detergent, and the other site (Stokes radius 9.6 nm, sedimentation coefficient 13.4 S) consists of 352 kDa protein and 569 kDa detergent. Frictional coefficients of these sites indicate that these receptor-detergent complexes are asymmetrical in structure, consistent with large transmembrane proteins.

V. EFFECTS OF THE ANTI-ANDROGEN TSAA-291 ON THE ANDROGEN-RECEPTOR COMPLEX FORMATION FROM [3H]TESTOSTERONE IN RAT VENTRAL PROSTATES

1979 ◽  
Vol 92 (3_Supplb) ◽  
pp. S67-S81 ◽  
Author(s):  
Katsuichiro Sudo ◽  
Keiji Yoshida ◽  
Yoshiaki Kimura ◽  
Ryo Nakayama
Keyword(s):  
Androgen Receptor ◽  
Gradient Centrifugation ◽  
Control Level ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Ventral Prostate ◽  
Sucrose Density Gradient Centrifugation ◽  
Macromolecular Complexes

ABSTRACT Intramuscular administration of a new steroidal anti-androgen, TSAA-291(16β-ethyl-17β-hydroxy-4-oestren-3-one), in doses of 0.05, 0.5 and 5 mg/kg body weight reduced the in vivo uptake of [3H]testosterone by the ventral prostate of castrated rats to 78, 59 and 37% of the control level, respectively. Analysis on subcellular fractions of the prostate by gel-filtration and sucrose density-gradient centrifugation followed by thin layer chromatographic identification of testosterone metabolites revealed that 5α-dihydrotestosterone(5α-DHT) which was found to be largely bound to macromolecules in the cytosol and nucleus was the predominant metabolite even in the presence of the anti-androgen, and radioactivities corresponding to the 5α-DHT-macromolecular complexes were decreased by the anti-androgen. TSAA-291 also inhibited the in vitro formation of the 5α-DHT-macromolecular complexes in both cytosol and nucleus from minced prostates incubated with [3H]testosterone. The importance of the findings is discussed in connection with the mode of anti-androgenic action of TSAA-291 in terms of the interaction with the androgen receptor.

scholarly journals Alteration of hepatic glucocorticoid receptor stability and nuclear binding in vitro by citrate

1983 ◽  
Vol 210 (1) ◽  
pp. 259-263 ◽  
Author(s):  
J Hubbard ◽  
M Kalimi
Keyword(s):  
Glucocorticoid Receptor ◽  
Glucocorticoid Receptors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Nuclear Binding ◽  
Wide Range

Citrate greatly stabilized rat hepatic unbound glucocorticoid receptors in cell-free conditions at 4 degrees C with optimal effectiveness at 5-15 mM. Control receptors were inactivated at 4 degrees C with a half-life of less than 12 h. However, in the presence of 10 mM-citrate, unbound receptors were almost completely stabilized for 48 h at 4 degrees C. Citrate at a concentration of 1-2 mM yielded half-maximal stabilization. The stabilizing effect of citrate was rather specific, as succinate, alpha-oxoglutarate, oxaloacetate, malate and pyruvate had no apparent stabilizing action. Citrate stabilized receptors over a wide range of H+ concentrations, with complete protection between pH 6.5 and 8.5. In addition, citrate appeared to have a significant effect on glucocorticoid-receptor complex activation into a nuclear binding form. Thus 5-10 mM-citrate enhanced nuclear binding, with optimal activation achieved at 10 mM concentration. As analysed by sucrose-density-gradient centrifugation and DEAE-cellulose chromatography, no apparent change was observed in the physical characteristics of the glucocorticoid receptor in the presence of citrate.

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