Deoxyribonucleic acid polymerase from nuclei of rat intestinal mucosa

1970 ◽  
Vol 48 (5) ◽  
pp. 529-536 ◽  
Author(s):  
F. Y. T. Leung ◽  
S. H. Zbarsky
Keyword(s):  
Dna Polymerase ◽  
Intestinal Mucosa ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Molecular Size ◽  
Sucrose Density Gradient ◽  
Polymerase Activity ◽  
Sucrose Density Gradient Centrifugation ◽  
Dna Template

An extract with DNA polymerase activity was prepared from nuclei of intestinal mucosa of the rat. Chromatography of the crude extract on DEAE-cellulose yielded three enzymically active fractions: I, II, and III. Each fraction could be resolved further into two components with DNA polymerase activity by rechromatography on smaller columns of DEAE-cellulose. A similar result was obtained by gel filtration of fraction II material on Sephadex G-150. The result of sucrose density gradient centrifugation of the fractions obtained by gel filtration suggested that each still consisted of a mixture of proteins with DNA polymerase activity. The approximate molecular weights of the active proteins, estimated by comparison with marker proteins, ranged from 25 000 to 300 000. Partially purified DNA polymerase (fraction II) required for activity the four deoxyribonucleoside triphosphates, Mg2+, 2-mercaptoethanol, and DNA template. The optimum pH for activity was 8.0 in Tris–HCl buffer and 7.4 in phosphate buffer. The two components obtained by gel filtration of fraction II differed in their requirements for DNA template. The one of smaller molecular size was more active with native DNA whereas the larger was equally active with either native or heat-denatured DNA.

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 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.

Enhancing Effect of Surfactant and Protein on Hydrolysis of Thymolphthalein Monophosphate by Purified Prostatic Acid Phosphatase

1975 ◽  
Vol 21 (12) ◽  
pp. 1761-1765 ◽  
Author(s):  
Andras G Foti ◽  
Harvey Herschman ◽  
J Fenimore Cooper ◽  
Hedi imFeld
Keyword(s):  
Acid Phosphatase ◽  
Serum Proteins ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Prostatic Acid Phosphatase ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Brij 35 ◽  
Hydrolysis Of ◽  
Protein Bovine Serum Albumin

Abstract Purified prostatic acid phosphatase catalyzes the hydrolysis of thymolphthalein monophosphate 10-fold faster if an optimal concentration of Brij 35 (a wetting agent) or protein (bovine serum albumin or human serum proteins) is present. Results of gel filtration, dialysis, and sucrose density-gradient centrifugation analysis suggest that the substrate must combine with detergent or protein before the enzyme can catalyze its hydrolysis.

FAMILIAL GOITRE WITH ABSENCE OF THYROGLOBULIN AND SYNTHESIS OF THYROID HORMONES FROM THYROIDAL ALBUMIN

1974 ◽  
Vol 60 (3) ◽  
pp. 389-397 ◽  
Author(s):  
K. B. DESAI ◽  
M. N. MEHTA ◽  
M. C. PATEL ◽  
S. M. SHARMA ◽  
L. RAMANNA ◽  
...  
Keyword(s):  
Thyroid Hormones ◽  
Radioactive Iodine ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Starch Gel Electrophoresis ◽  
Sucrose Density Gradient Centrifugation ◽  
Salting Out ◽  
Immunological Tests ◽  
Starch Gel

SUMMARY Two siblings, a brother (H. B.) and a sister (R. B.) with long standing goitres were investigated. Radioactive iodine uptake by the thyroid was increased and a significant portion of the plasma radioactive iodine was not extractable with butanol. Chromatography of butanol extracts of serum after radioactive iodine administration showed distinct peaks of triiodothyronine and thyroxine. Microscopic examination of the surgical specimens of the goitres showed Hürthle cell carcinoma with follicles devoid of colloid in both specimens. Sucrose density gradient centrifugation, gel filtration on Sephadex G-200, salting out procedures, starch gel electrophoresis and immunological tests of the supernatant soluble fraction of thyroid homogenates showed a lack of thyroglobulin. Further fractionation of the soluble proteins showed that albumin was apparently involved in the synthesis of thyroid hormones in the absence of thyroglobulin.

scholarly journals Molecular size of the 5-HT3 receptor solubilized from NCB 20 cells

1990 ◽  
Vol 269 (3) ◽  
pp. 623-628 ◽  
Author(s):  
R M McKernan ◽  
C S Biggs ◽  
N Gillard ◽  
K Quirk ◽  
C I Ragan
Keyword(s):  
Physical Properties ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Receptor Site ◽  
Molecular Size ◽  
Sucrose Density Gradient ◽  
Partial Specific Volume ◽  
Sucrose Density Gradient Centrifugation ◽  
Ics 205 930

The 5-HT3 hydroxytryptamine receptor from NCB 20 cells was solubilized and the molecular and hydrodynamic properties of the receptor were investigated. The receptor was identified by binding of the radioligand 3-NN′-[3H]dimethyl-8-azabicyclo[3.2.1]octanyl indol-3-yl carboxylate ester [(3H]Q ICS 205-930) to NCB 20 membranes (Bmax = 1.19 +/- 0.31 pmol/mg of protein; Kd = 0.43 +/- 0.076 nM) and was optimally solubilized with 0.5% deoxycholate. [3H]Q ICS 205-930 labelled one population of sites in solution (Bmax = 1.11 +/- 0.4 pmol/mg of protein; Kd = 0.48 +/- 0.06 nM; n = 4). The characteristics of [3H]Q ICS 205-930 binding were essentially unchanged by solubilization, and competition for [3H]Q ICS 205-930 binding by a series of 5-HT3 agonists and antagonists was consistent with binding to a 5-HT3 receptor site and was similar to that observed for 5-HT3 receptors solubilized from rat brain [McKernan, Quirk, Jackson & Ragan (1990) J. Neurochem. 54, 924-930]. Some physical properties of the solubilized receptor were investigated. The molecular size (Stokes radius) of the [3H]Q ICS 205-930-binding site was measured by gel-exclusion chromatography in a buffer containing 0.2% Lubrol and 0.5 M-NaCl and was determined as 4.81 +/- 0.15 nm (mean +/- S.E.M.; n = 6). Sucrose-density-gradient centrifugation was also performed under the same detergent and salt conditions to determine the partial specific volume (v) of the detergent-receptor site complex. This was found to be 0.794 ml.g-1. Sucrose-density-gradient centrifugation was carried out in both 1H2O and 2H2O to allow correction for detergent binding to the receptor. The Mr of the 5-HT3 receptor under these conditions was calculated as 249,000 +/- 18,000 (n = 3). The size and physical properties of the 5-HT3 receptor are similar to those observed for members of the family of ligand-gated ion channels.

scholarly journals Purification of the enzyme NADPH: protochlorophyllide oxidoreductase

1981 ◽  
Vol 195 (1) ◽  
pp. 83-92 ◽  
Author(s):  
N S Beer ◽  
W T Griffiths
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton X ◽  
Etioplast Membranes

A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.

Seminiferous tubule androgen receptors in experimental cryptorchidism

1986 ◽  
Vol 109 (3) ◽  
pp. 427-433
Author(s):  
S. J. Winters
Keyword(s):  
Receptor Binding ◽  
Seminiferous Tubule ◽  
Androgen Receptors ◽  
Binding Capacity ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Vertical Tube ◽  
Testis Weight ◽  
Sucrose Density Gradient Centrifugation

ABSTRACT As an initial approach to the study of seminiferous tubule androgen receptors in disordered spermatogenesis, cytosol androgen receptors were studied in rats with experimental cryptorchidism. Two weeks after the testis had been repositioned in the abdomen of 6-week-old rats, the animals were hypophysectomized to deplete the testis of androgen, and 1 week later they were killed. Androgen receptor binding was studied in seminiferous tubule cytosol using [3H]methyltrienolone as the radiolabelled probe. The androgen-binding capacity of cryptorchid testis, when expressed as fmol bound/testis, was reduced to 50% of control, in parallel with the decline in testis weight. No change in binding affinity was found. Sucrose density gradient centrifugation using a vertical tube rotor revealed a 9S molybdate-stabilized receptor under low-salt conditions in both cryptorchid and scrotal seminiferous tubule cytosol. Receptor-complex stability studies, analysis by gel filtration and DEAEcellulose chromatography produced similar results in cryptorchid and scrotal tubules. The mechanism for the reduction in testicular receptor content of an abdominal testis remains to be clarified. The demonstration that testicular androgen receptors can be reduced by cryptorchidism suggests that further studies may indicate the role of receptor binding in testicular function. J. Endocr. (1986) 109, 427–433

Analysis of activated androgen receptors in rat brain and anterior pituitary and ventral prostate glands: nuclear binding and RNA polymerase activity

1984 ◽  
Vol 102 (2) ◽  
pp. 181-188 ◽  
Author(s):  
B. D. Greenstein
Keyword(s):  
Rna Polymerase ◽  
Anterior Pituitary ◽  
Androgen Receptors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Polymerase Activity ◽  
Ventral Prostate ◽  
Sucrose Density Gradient Centrifugation ◽  
Rna Polymerase Activity

ABSTRACT Cytosolic androgen receptors from neocortex, hypothalamus and anterior pituitary and ventral prostate glands were analysed by miniature isoelectric focusing and sucrose density-gradient centrifugation before and after precipitation of [3H]dihydrotestosterone (DHT)-bound complexes with ammonium sulphate. In the hypothalamus and neocortex (NH4)2SO4 precipitation appeared to cause heterodisperse peaks, and in the case of the ventral prostate there was a clear shift to a more basic isoelectric point. After sucrose density-gradient centrifugation all cytosols sedimented as large aggregates which appeared to dissociate into subunits in 0·4 m-KCl gradients. The functional significance of these altered forms was tested by nuclear uptake studies of cytosolic [3H]DHT-bound complexes, which could only be detected in brain and pituitary nuclei after prior precipitation with (NH4)2SO4, which also significantly increased extraction of ventral prostate [3H]DHT-bound complexes from the nucleus. The nuclei apparently responded to the (NH4)2SO4-precipitated and redissolved complexes by increased RNA polymerase activity. These results are consistent with the possibility that the neural androgen receptor is altered before interaction with the genome, and this alteration may be necessary for the action of the hormone to be expressed. J. Endocr. (1984) 102, 181–188

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