scholarly journals The effect of whole-body X-irradiation of guinea pigs on liver ribonucleic acid synthesis

1970 ◽  
Vol 116 (3) ◽  
pp. 503-514 ◽  
Author(s):  
E. J. Hidvégi ◽  
Elisabeth Bölöni ◽  
J. Holland ◽  
F. Antoni ◽  
V. Várterész
Keyword(s):  
Orotic Acid ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Polymerase Activity ◽  
Whole Body ◽  
Sucrose Density Gradient Centrifugation ◽  
Polyuridylic Acid ◽  
X Irradiation ◽  
Rna Polymerase Activity

1. Liver RNA synthesis was studied within 24h after whole-body X-irradiation of guinea pigs that had been starved for 22–24h. 2. Microsomal RNA was labelled in vivo for 3h with [14C]orotic acid and the isolated labelled RNA was fractionated by sucrose-density-gradient centrifugation. Incorporation was 50–100% higher between 3 and 12h after 2000rd X-irradiation and at 22h was not elevated any further. Whole nuclear RNA was labelled with [14C]orotic acid for 15min. At 5h after irradiation the incorporation showed a 50–100% increase. Incorporation increased in all types of RNA studied. 3. The RNA phosphorus/DNA phosphorus ratio of whole liver gradually increased after X-irradiation. Maximal increase was found between 24 and 36h, which corresponds to a value about 40% above that of the starved control. The RNA phosphorus content of isolated ribonucleoproteins obtained from various cell fractions of the liver was similarly increased after X-irradiation. 4. Liver microsomes were obtained from X-irradiated and control animals. Microsomes were incubated in vitro with [14C]phenylalanine in the presence and absence of polyuridylic acid. After the incubation the microsomes were fractionated by sucrose-density-gradient centrifugation. The polyuridylic acid enhancement was twice as great in the microsomes of the control preparation as in the irradiated one. The experiment demonstrated a higher saturation of microsomes by endogenous messenger after X-irradiation. 5. RNA polymerase activity of the purified nuclear preparation was assayed. The activity of the Mg2+-dependent RNA polymerase activity was 50 and 200% respectively above the control values at 6 and 9h after X-irradiation. 6. Animals were treated with actinomycin D shortly before X-irradiation. This treatment abolished the radiation-induced enrichment of polyribosomes and the increase of protein-synthesizing activity. The effect of X-irradiation on the transcription of the genetic code of the liver is discussed.

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

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

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.

3 ß-Hydroxysteroid Oxidoreductase in Suspension Cultures of Digitalis lanata EH RH

1990 ◽  
Vol 45 (9-10) ◽  
pp. 963-972 ◽  
Author(s):  
Hildegard Maria Warneck ◽  
Hanns Ulrich Seitz
Keyword(s):  
Incubation Temperature ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Cell Compartment ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Digitalis Lanata ◽  
Substrate Kinetics ◽  
Soluble Part

Abstract A 3 β-hydroxysteroid oxidoreductase was isolated and characterized in the microsomes of Digitalis lanata cell cultures. The enzyme catalyzes the conversion of 5α-pregnane-3,20-dione to 5a-pregnan-3 β-ol-20-one and requires NAD(P)H2. The enzyme was found to have a pH optimum of 80. The reaction had an optimum incubation temperature of 25 °C with linear reduction for the first 4 h, reaching maximum enzyme activity after 7 h. Substrate kinetics for 5a-pregnane-3,20-dione and NADPH2 resulted in apparent Km-values of 18.5-20 (µM for 5a-pregnane-3,20-dione and 50-120 µM for the co-substrate NADPH2. In order to localize 3β-hydroxysteroid oxidoreductase differential centrifugation as well as linear sucrose density gradient centrifugation were performed. The results obtained lead to the conclusion that 3β-hydroxysteroid oxidoreductase is not associated with a single cell compartment, but consists of a major soluble part and a markedly smaller part of endoplasmic reticulum-associated activity

scholarly journals Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins

1980 ◽  
Vol 185 (3) ◽  
pp. 667-677 ◽  
Author(s):  
J Elliott ◽  
S G Blanchard ◽  
W Wu ◽  
J Miller ◽  
C D Strader ◽  
...  
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Selective Extraction ◽  
Synaptic Membranes ◽  
Detergent Solution ◽  
Major Step ◽  
Sucrose Density Gradient Centrifugation ◽  
Torpedo Californica

A rapid methof for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-idosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures.

DEMONSTRATION OF A CYTOPLASMIC RECEPTOR PROTEIN FOR OESTROGEN IN THE CANINE PROSTATE GLAND

1978 ◽  
Vol 78 (1) ◽  
pp. 131-139 ◽  
Author(s):  
N. CHAISIRI ◽  
Y. VALOTAIRE ◽  
BRONWEN A. J. EVANS ◽  
C. G. PIERREPOINT
Keyword(s):  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Prostate Gland ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Receptor Protein ◽  
Receptor Complex ◽  
Binding Properties ◽  
Sucrose Density Gradient Centrifugation ◽  
A Value

A receptor protein that selectively binds oestrogens has been demonstrated in the cytosol of the canine prostate gland. The steroid–receptor complex was found to have a sedimentation coefficient of 4–5 S with respect to bovine serum albumin after sucrose density-gradient centrifugation. The high affinity and low capacity of the protein for oestrogens was indicated by displacement studies, which gave a value of 3·8 ± 1·53 (s.d.) × 10−10 mol/l for the dissociation constant. A metastasizing prostatic tumour was also shown to possess this receptor, with binding properties similar to those exhibited by the receptor in normal prostatic cytosol. The implications of these findings are discussed with regard to normal prostatic function in the dog and the virtually inevitable advent of prostatic hyperplasia with age in this species.

Sign in / Sign up

Export Citation Format

Share Document