scholarly journals A chromosomal phosphoprotein is preferentially released by mild micrococcal-nuclease digestion

1984 ◽  
Vol 220 (2) ◽  
pp. 539-545 ◽  
Author(s):  
C C Liew ◽  
M J Halikowski ◽  
M S Zhao
Keyword(s):  
Gradient Centrifugation ◽  
Specific Radioactivity ◽  
Sucrose Density Gradient ◽  
Micrococcal Nuclease ◽  
Chromosomal Protein ◽  
Sucrose Density Gradient Centrifugation ◽  
Micrococcal Nuclease Digestion ◽  
Nuclease Digestion ◽  
32P Incorporation ◽  
Liver Nuclei

[32P]Pi was administered to rats (5mCi/rat) 2h before the isolation of liver nuclei. The isolated nuclei were subjected to mild micrococcal-nuclease digestion for 2.5, 5 and 10 min at 37 degrees C, and the mononucleosomal fraction was subsequently isolated by sucrose-density-gradient centrifugation. The specific radioactivity of 32P-labelled mononucleosomal fractions decreased with increased digestion times. A phosphorylated chromosomal protein, B2 (Mr 68000, pI6.5-8.2), was demonstrated immunologically in the mononucleosomal fraction by using an antibody specific to this electrophoretically purified phosphoprotein. The incorporation of 32P into this phosphoprotein, previously shown to be mainly through covalent linkage, was revealed by antibody precipitation followed by gel electrophoresis. The rate of release of acid-soluble nucleotides by micrococcal-nuclease digestion of liver nuclei from partially hepatectomized rats 16 h after operation was strikingly higher than that for sham-operated controls. After partial hepatectomy, an increase in 32P incorporation into phosphoprotein in the monomer fractions specifically precipitated by this antibody was also found. This suggests that the phosphorylated non-histone chromatin protein B2 is preferentially associated with the transcriptionally active chromatin.

scholarly journals Localization of ferrochelatase and of newly synthesized haem in membrane fractions from Rhodopseudomonas spheroides

1978 ◽  
Vol 174 (1) ◽  
pp. 277-281 ◽  
Author(s):  
J Barrett ◽  
O T G Jones
Keyword(s):  
Cell Envelope ◽  
Gradient Centrifugation ◽  
Specific Radioactivity ◽  
Sucrose Density Gradient ◽  
Particulate Fraction ◽  
Outer Face ◽  
Sucrose Density Gradient Centrifugation ◽  
Intact Cells ◽  
Rhodopseudomonas Spheroides ◽  
Cytochrome Content

Cells of Rhodopseudomonas spheroides, strains R-26 or GVP, were grown photosynthetically, disrupted and two particulate fractions separated by sucrose-density-gradient centrifugation. The upper particulate fraction, enriched in bacteriochlorophyll, was identified as containing the chromatophores; the lower particulate fraction had the characteristics of the cell envelope. The two fractions differed in cytochrome content and cytochrome spectra. Ferrochelatase was found almost exclusively in the chromatophore fraction and was located on the outer face of the chromatophores, i.e. in contact with the cytosol in intact cells. The addition of 59FeCl3 to cells growing in low-iron media resulted in labelling of the protohaem fraction (probably arising from cytochrome b) of the membranes. The specific radioactivity of the haem of the chromatophores rose more rapidly than that of the envelope fraction and then after 2 h declined to approximately the same value, suggesting that haems of the chromatophore may act as precursors of haem of the envelope.

scholarly journals Mechanism of enhanced RNA synthesis in acute-phase rat liver and its relationship to chromatin structure

1984 ◽  
Vol 219 (1) ◽  
pp. 165-171 ◽  
Author(s):  
L Schiaffonati ◽  
L Bardella ◽  
G Cairo ◽  
V Giancotti ◽  
A Bernelli-Zazzera
Keyword(s):  
Acute Phase ◽  
Chromatin Structure ◽  
Rna Synthesis ◽  
Elongation Rate ◽  
Micrococcal Nuclease ◽  
Chromosomal Proteins ◽  
Micrococcal Nuclease Digestion ◽  
Nuclease Digestion ◽  
Liver Nuclei ◽  
Phase Process

Nuclei isolated from the liver of rats undergoing an acute inflammatory reaction induced by turpentine treatment show increased RNA synthesis. This increase is essentially determined by a faster polyribonucleotide-elongation rate while the number of transcribing polymerase molecules is unchanged. The sensitivity of chromatin to micrococcal-nuclease digestion and the composition of chromosomal proteins are not affected by the acute-phase process. Therefore the increased RNA synthesis by liver nuclei from acutely inflamed rats does not seem to correlate with major changes in chromatin structure.

scholarly journals Nuclear association states of rat uterine oestrogen receptors as probed by nuclease digestion

1981 ◽  
Vol 196 (2) ◽  
pp. 423-432 ◽  
Author(s):  
Daniel R. Schoenberg ◽  
James H. Clark
Keyword(s):  
Time Course ◽  
Gradient Centrifugation ◽  
Female Rats ◽  
Micrococcal Nuclease ◽  
Oestrogen Receptors ◽  
Sucrose Density Gradient Centrifugation ◽  
Dna Hydrolysis ◽  
Oestradiol Treatment ◽  
Nuclease Digestion ◽  
Receptor Solubilization

The solubilization of oestrogen receptors from uterine nuclei by micrococcal nuclease and deoxyribonuclease I was examined after the injection of oestradiol or Nafoxidine into castrated female rats. At 1h after an injection of oestradiol, 30% (0.18pmol/mg of DNA) of the nuclear oestrogen receptors was solubilized by 5 min of mild digestion with either nuclease. No further receptor release occurred, although DNA hydrolysis continued throughout a 20min interval. The limitation in receptor solubilization was not due to an artifact of digestion conditions or insufficient nuclease concentrations. Similar patterns of receptor solubilization and DNA hydrolysis were obtained with both nucleases whether the animals had been injected with oestradiol 1h before death or if the uteri from uninjected animals were incubated with [3H]oestradiol for 1h in vitro. When uterine nuclei were digested with these enzymes 12h after the animal was injected with oestradiol there was little change in the quantity of nuclease-sensitive sites (0.11pmol/mg of DNA); however, the quantity of nuclease-resistant sites decreased 10-fold. These values correspond quantitatively to the changes in salt-resistant and salt-extractable sites observed over a 12h interval after oestradiol treatment. Nuclease digestion of uterine nuclei obtained 16h after Nafoxidine treatment gave a pattern qualitatively and quantitatively similar to that observed 1h after oestradiol treatment, a result consistent with the agonist/antagonist action of this compound. An analysis by sucrose-density-gradient centrifugation of the time course of nuclease-dependent receptor solubilization indicated that the solubilized receptors were not associated with discrete nucleosomal fragments. We believe that these data indicate that only a portion of the receptors translocated to the nucleus become associated with chromatin, and this association may occur on regions of chromatin that are preferentially susceptible to nucleolytic cleavage.

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 Molecular heterogeneity of mouse duodenal alkaline phosphatase. Association of lipids and peptides

1974 ◽  
Vol 141 (1) ◽  
pp. 93-101 ◽  
Author(s):  
P. R. V. Nayudu ◽  
Fraser B. Hercus
Keyword(s):  
Alkaline Phosphatase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Molecular Forms ◽  
Partial Specific Volume ◽  
Molecular Heterogeneity ◽  
Sucrose Density Gradient Centrifugation

Polyacrylamide-gel electrophoresis and Bio-Gel P-300 molecular-sieve chromatography of mouse duodenal alkaline phosphatase demonstrates its molecular heterogeneity, which, in a kinetic sense, is manifest also in the differential relative velocities of the heterogeneous forms of the enzyme with two substrates, phenylphosphate and β-glycerophosphate. Different treatments that eliminate most of the electrophoretic and chromatographic variability of the enzyme also decrease the velocities with both substrates so that the molar ratio of hydrolysis of one substrate relative to the other is also altered to a low but stable value. Concomitant with these changes, lipids and peptides are dissociated from the enzyme. The lipids are tentatively identified as a sterol and phospholipids. The peptides have an average composition of four to six amino acids and appear to be strongly electropositive. The conditions of dissociation suggest that their binding to the enzyme is non-covalent and predominantly based on hydrophobic and ionic bonding. The concept of lipid and peptide association would suggest prima facie differential molecular weights as a factor in the observed electrophoretic and chromatographic heterogeneity. However, the molecular forms of the enzyme with differences in elution volume equivalent to more than one-half the void volume of the Bio-Gel P-300 column, or even enzyme fractions dissociated from the lipids and peptides compared with undissociated portions, do not show any differences in sedimentation on sucrose-density-gradient centrifugation. This may be because the alterations in molecular weight owing to binding of small molecules are too small to be detected by this method. Alternatively, since lipids are involved, the binding may alter the partial specific volume in such a way that the buoyant density is not significantly altered.

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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