scholarly journals SOLUBLE ENZYMES OF NUCLEI ISOLATED IN SUCROSE AND NON-AQUEOUS MEDIA

1953 ◽  
Vol 37 (2) ◽  
pp. 177-187 ◽  
Author(s):  
Herbert Stern ◽  
A. E. Mirsky
Keyword(s):  
Adenosine Deaminase ◽  
Organic Solvents ◽  
Rat Liver ◽  
Nuclear Membrane ◽  
Aqueous Media ◽  
Isolated Nuclei ◽  
Calf Thymus ◽  
Liver Nuclei ◽  
Soluble Enzymes ◽  
Soluble Components

Nuclei of calf thymus and liver and of rat liver were isolated in sucrose media and a number of their properties studied in relation to those of corresponding nuclei isolated in non-aqueous media with a view to determining their capacity to retain soluble components. The best preparations of sucrose nuclei were obtained from calf thymus. Cytochrome oxidase measurements and DNA/N ratios were far less sensitive than microscopic examination as indicators of purity when rat liver and calf thymus nuclei were compared. No satisfactory preparation of calf liver nuclei was obtained, contamination with whole cells having been appreciable; such preparations, nevertheless, could be used to advantage in the tests undertaken. DNA content of thymus nuclei isolated in sucrose was much the same as that of non-aqueous ones, pointing to a retention of soluble protein under aqueous conditions of isolation. That this net retention of protein was not due to the impermeability of the nuclear membrane was shown by the hydrolysis of the DNA upon addition of some crystalline DNAase to a sucrose suspension of nuclei. A comparative study of liver and thymus nuclei isolated in aqueous and non-aqueous media with respect to the soluble enzymes glucose-6-phosphate dehydrogenase, adenosine deaminase, and nucleoside phosphorylase yielded the following results: 1. Lyophilization of sucrose-isolated nuclei and their extraction with the organic solvents used in the non-aqueous procedure did not inactivate any of the enzymes tested. In the case of thymus the reverse was true, there being a marked increase in activity of all the enzymes studied. 2. In thymus, nucleoside phosphorylase and adenosine deaminase were active to approximately the same extent in nuclei isolated by either procedure. Glucose phosphate dehydrogenase alone was more active in sucrose-isolated nuclei, pointing to the possibility of an adsorption of this enzyme. 3. In rat liver nuclei isolated in sucrose, lyophilization and treatment with organic solvents revealed only the presence of some dehydrogenase. 4. The washing out of soluble enzymes was most markedly demonstrated in the case of calf liver. Only traces of the nucleoside enzymes were found in the sucrose-isolated nuclei, and in the case of the dehydrogenase only a half of that present in the non-aqueous nucleus remained. The main conclusions drawn were as follows:— 1. In sucrose media the nuclear membrane is ineffectual in preventing the inward or outward diffusion of protein. 2. The extent to which soluble proteins are retained by a nucleus isolated in sucrose appears to depend upon internal structural factors, such as the concentration of DNA in the nucleus. 3. With respect to determining the composition of nuclei in terms of soluble components, the sucrose isolation procedure is considered to be of indifferent merit and hence invalid for such a type of analysis.

Dissociation of the triiodothyronine receptor from the nucleus induced by cations: evidence for divalent cation sensitive and insensitive nuclear sites

1985 ◽  
Vol 110 (4) ◽  
pp. 510-514 ◽  
Author(s):  
Juan Bernal ◽  
Ana Perez-Castillo
Keyword(s):  
Rat Liver ◽  
Divalent Cation ◽  
Nuclear Membrane ◽  
Second Phase ◽  
Rat Liver Nuclei ◽  
Liver Nuclei ◽  
Fast Release

Abstract. Aliquots of purified rat liver nuclei were diluted at 0°C in isotonic buffers containing monovalent (Na+) or divalent (Ca2, Mg2+) cations. At different times following dilution the nuclear suspensions were centrifuged and the T3 receptor was measured in KCl extracts of the nuclear pellets. The rate of receptor loss after dilution in EDTA was 0.0025 min−1. Dilution in the presence of cations caused a fast release of receptor during the first 10 min. This phase, which was not observed when the nuclei were diluted in EDTA without salt, was followed by a second phase where the receptor was released at the same rate as in EDTA. Receptor release was only dependent on the presence of cations in the dilution buffer during the first 10 min after dilution. The amounts of receptor remaining in the nuclei after the first 10 min of dilution were 51.8 ± 9.2%, in the presence of Ca2+and Mg2+, 38.6 ±8.9% in 0.15 m NaCl, and 18.0 ± 4.8% in 0.15 m NaCl in the presence of Ca2+ and Mg2+. The release of receptor was not influenced by the integrity of the nuclear membrane. These results suggest the presence of divalent cation sensitive and insensitive nuclear sites for the T3 receptor, in amounts which could be estimated to be about 48 and 52%, respectively. Other interpretations are also possible, such as the presence of a high proportion of free receptors in the nucleosol, which could be released during the first phase of dilution if the negative charges in chromatin are blocked by cations to avoid redistribution of receptors immediately after dilution.

scholarly journals Synthesis of polyphosphoinositides in nuclei of Friend cells. Evidence for polyphosphoinositide metabolism inside the nucleus which changes with cell differentiation

1987 ◽  
Vol 248 (3) ◽  
pp. 765-770 ◽  
Author(s):  
L Cocco ◽  
R S Gilmour ◽  
A Ognibene ◽  
A J Letcher ◽  
F A Manzoli ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Phosphatidic Acid ◽  
Nuclear Membrane ◽  
Isolated Nuclei ◽  
Phosphatidylinositol Bisphosphate ◽  
Phosphatidylinositol Kinase ◽  
Phosphatidylinositol Phosphate ◽  
Friend Cells ◽  
Liver Nuclei ◽  
Polyphosphoinositide Metabolism

Previous work demonstrated the existence of phosphatidylinositol kinase and phosphatidylinositol phosphate kinase in rat liver nuclei, with the suggestion that these activities are in the nuclear membrane [Smith & Wells (1983) J. Biol. Chem. 258, 9368-9373]. Here we show that highly purified nuclei from Friend cells, washed free of nuclear membrane by Triton, can incorporate radiolabel from [gamma-32P]ATP into phosphatidic acid, phosphatidylinositol phosphate and phosphatidylinositol 4,5-bisphosphate. The degree of radiolabelling of phosphatidylinositol bisphosphate is highly dependent on the state of differentiation of the cells, being barely detectable in growing cells and much greater after dimethyl sulphoxide-induced differentiation; this difference is mostly due to different amounts of phosphatidylinositol phosphate in the isolated nuclei. We suggest that polyphosphoinositides are made inside the nucleus and that they have a role in chromatin function; either the phospholipids themselves play a role, or there is a possibility of intranuclear signalling by inositide-derived molecules.

scholarly journals Effects of polyamines and methylglyoxal bis(guanylhydrazone) on hepatic nuclear structure and deoxyribonucleic acid template activity

1975 ◽  
Vol 151 (3) ◽  
pp. 505-512 ◽  
Author(s):  
K B Brown ◽  
N F Nelson ◽  
D G Brown
Keyword(s):  
Dna Polymerase ◽  
Rat Liver ◽  
Template Activity ◽  
Isolated Rat Liver ◽  
E Coli ◽  
Rat Liver Nuclei ◽  
Calf Thymus ◽  
Liver Nuclei ◽  
Dna Template ◽  
Isolated Rat

1. The interaction of polyamines and methylglyoxal bis(guanythydrazone) (1, 1′-[(methylethanediylidene)-dinitrilo]diguanidine) with isolated rat liver nuclei was investigated by electron microscopy. 2. At 4mM, putrescine was without effect; however, spermidine, spermine or methylglyoxal bis(guanythydrazone) resulted in dispersed chromatin and alterations in nucleolar structure. In addition, spermidine or methylglyoxal bis(guanylhydrazone) caused marked aggregation of interchromatin granules. 3. The DNA template property of calf thymus DNA was examined by using DNA polymerases from Escherichia coli, Micrococcus lysodeikticus and calf thymus in the presence of 0-5 mM-amine. 4. In the presence of DNA polymerase, spermine or methylglyoxal bis(guanylhydrazone) inhibited activity, whereas putrescine or spermidine had much less effect or in some cases stimulated [3H]dTMP incorporation. 5. Template activity which was inhibited by spermine or methylglyoxal bis(guanylhydrazone) could be partially restored by additional DNA or enzyme. 6. When mixed with calf thymus DNA, calf thymus histone inhibited template activity as measured with E. coli DNA polymerase. The template activity of such a ‘histone-nucleate’ could not be restored by putrescine, spermidine, spermine or methylglyoxal bis(guanylhydrazone). 7. DNA template activity of isolated rat liver nuclei was tested by using E. coli DNA polymerase. None of the amines was able to increase the template activity of the nuclear DNA in vitro.

scholarly journals STUDIES OF TWO TYPES OF ALKALINE PHOSPHATASE IN NUCLEI ISOLATED FROM THE LIVERS OF FED AND FASTED RATS BY A MODIFICATION OF THE BEHRENS TECHNIQUE

1955 ◽  
Vol 1 (4) ◽  
pp. 331-338 ◽  
Author(s):  
Arthur J. Emery ◽  
Alexander L. Dounce
Keyword(s):  
Alkaline Phosphatase ◽  
Rat Liver ◽  
Liver Cell ◽  
Aqueous Media ◽  
Slight Modification ◽  
Magnesium Ions ◽  
Rat Liver Nuclei ◽  
Liver Nuclei ◽  
The One ◽  
Fasted Rats

1. Rat liver nuclei were isolated from normal rats and rats fasted for 36 hours by a slight modification of the Behrens technique. 2. The nucleus of the rat liver cell contains two types of alkaline phosphatase. This confirms the previous findings on rat liver nuclei isolated in aqueous media. 3. The one type of alkaline phosphatase is not activated by magnesium ions, and this enzyme is very strongly bound to structural material of the nucleus. The other type of alkaline phosphatase is activated by magnesium ions, and this enzyme is probably free to diffuse from cytoplasm to nucleus and vice versa through the nuclear membrane. 4. Fasting caused a pronounced decrease of protein in general and of the alkaline phosphatase which is activated by magnesium ions from the nucleus of the rat liver cell, while the alkaline phosphatase that is not activated by magnesium was less affected.

Detection of 5-Hydroxymethyl-2′-Deoxyuridine in Dna from Calf Thymus, Hela Cells, and Rat Liver Nuclei

1990 ◽  
Vol 9 (2) ◽  
pp. 179-187 ◽  
Author(s):  
Russell C. Cattley ◽  
Susan R. Dietze ◽  
Frank C. Richardson ◽  
James A. Popp
Keyword(s):  
Rat Liver ◽  
Hela Cells ◽  
Rat Liver Nuclei ◽  
Calf Thymus ◽  
Liver Nuclei

Two Forms of Histone–Acetyltransferase in High Salt Extracts of Rat Liver Nuclei

1973 ◽  
Vol 51 (8) ◽  
pp. 1177-1194 ◽  
Author(s):  
Peter F. Lue ◽  
A. G. Gornall ◽  
C. C. Liew
Keyword(s):  
Rat Liver ◽  
Histone Acetyltransferase ◽  
Extraction Procedure ◽  
Gel Filtration ◽  
Electrophoretic Analysis ◽  
High Salt ◽  
Sephadex Column ◽  
Nuclear Extracts ◽  
Calf Thymus ◽  
Liver Nuclei

Two forms of 'histone' acetyltransferase (AT I and AT II) were separated by DEAE-Sephadex chromatography of rat liver nuclear extracts prepared by sonication in 0.3 M (NH4)2SO4. A 12-fold greater amount of AT I was not adsorbed on the DEAE-Sephadex column, whereas AT II was adsorbed and was eluted with 0.15–0.2 M (NH4)2SO4. Chromatography of AT I on Sephadex G-200 and cellulose phosphate indicated the presence of additional forms of the enzyme. Optimal conditions for the assay of the enzyme have been determined.In vitro acetylation of calf thymus histone fractions by AT I or AT II showed that at low histone concentration (313 μg/ml), the "arginine-rich" fractions (f2a1 and f3) were acetylated to a greater extent than were the "lysine-rich" fractions (f1 and f2b). However, at a higher concentration of histone (1250 μg/ml) f2a1 acetylation decreased as a result of coprecipitation with the enzyme. Electrophoretic analysis of the acetylated fractions indicated that multiple sites were probably acetylated on f3, f2a2, and f2a1, whereas only one residue was reactive in f1 and f2b.Interconversion of AT I and AT II has been demonstrated and it was shown that [Formula: see text]. AT I contained mainly histones f2a2 and f3, and was converted to AT II by treatment with high salt and gel filtration chromatography. After hypotonic lysis of nuclei and DEAE chromatographic analysis of extracts obtained by washing the nuclear pellet with 0.3 M sodium citrate of NaCl, most of the acetylating activity in these extracts was identified as AT II. Thus the greater amount of AT I observed initially in (NH4)2SO4 extracts was probably a consequence of the sonication step used in this extraction procedure.

scholarly journals Metabolism and possible compartmentalization of inositol lipids in isolated rat-liver nuclei

1997 ◽  
Vol 327 (2) ◽  
pp. 569-576 ◽  
Author(s):  
R. Lewis VANN ◽  
Peter F. B. WOODING ◽  
F. Robin IRVINE ◽  
Nullin DIVECHA
Keyword(s):  
Phosphatidic Acid ◽  
Rat Liver ◽  
Nuclear Membrane ◽  
Initial Increase ◽  
Similar Amount ◽  
Isolated Rat Liver ◽  
Rat Liver Nuclei ◽  
Inositol Lipids ◽  
Liver Nuclei ◽  
Isolated Rat

(1) The removal of the nuclear envelope from isolated rat-liver nuclei by washing with Triton X-100 (TX-100) was assessed by electron microscopy. All the envelope was removed by 0.04% (w/v) TX-100. (2) After this removal, phosphorylation of inositol lipids and diacylglycerol (DAG) from [γ-32P]ATP still occurs, despite the near complete absence of detectable (by mass assay) DAG and PtdIns. This suggests that the majority of these two lipids in nuclei are present in the nuclear membrane, but the small amounts remaining after extraction, defined as intranuclear, are available for phosphorylation by lipid kinases (36% for DAG and 24% for PtdIns respectively, when expressed as a percentage of incorporation of intact nuclei). (3) PtdIns(4,5)P2 did not follow the same pattern as PtdIns and DAG; after removal of the nuclear membrane, 40% of the mass of this lipid was left in the nucleus. Moreover, a similar amount of PtdIns(4,5)P2 was also resistant to extraction with even higher concentrations of detergent, suggesting that PtdIns(4,5)P2 has a discrete intranuclear location, probably bound to nuclear proteins. (4) Addition of exogenous substrates, PtdIns, PtdIns(4)P and DAG, to membrane-depleted nuclei resulted in reconstitution of the majority of lipid phosphorylations from [γ-32P]ATP (70%, 90% and 94% of intact nuclei respectively), suggesting a predominantly intranuclear location for the respective kinases. (5) Nuclei also showed phosphomonoesterase and phosphatidic acid hydrolase activity; dephosphorylation of pre-radiolabelled PtdIns(4)P, PtdIns(4,5)P2 and phosphatidic acid was observed when [γ-32P]ATP was removed. However, some of the radioactivity was apparently resistant to these enzymes, suggesting the existence of multiple pools of these lipids. (6) Addition of excess non-radiolabelled ATP to nuclei pre-labelled with [γ-32P]ATP resulted in an initial increase in the label in PtdIns(4,5)P2, implying a precursor-product relationship between the radiolabelled pools of PtdIns(4)P and PtdIns(4,5)P2. This was confirmed by analysis of the incorporation of 32P into the 4ʹ-phosphate group of PtdIns(4)P and the individual 4ʹ- and 5ʹ-phosphate groups of PtdIns(4,5)P2. The data from these experiments also indicated that PtdIns(4,5)P2 can be produced from a pre-existing pool of PtdIns(4)P, as well as de novo from PtdIns. (7) Taken together our data suggest that isolated rat-liver nuclei have an intranuclear inositol lipid metabolism mechanism utilizing enzymes and substrates equivalent to those found in cytosol and plasma membrane, and that there may be some, but not complete, compartmentalization of the components of the nuclear inositol cycle.

scholarly journals High total histone/deoxyribonucleic acid ratios for rat liver nuclei

1973 ◽  
Vol 135 (1) ◽  
pp. 115-123 ◽  
Author(s):  
Subir K. Chanda ◽  
Regina Ickowicz ◽  
Alexander L. Dounce
Keyword(s):  
Amino Acid ◽  
Direct Measurement ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Deoxyribonucleic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Total Histone ◽  
Rat Liver Nuclei ◽  
Calf Thymus ◽  
Liver Nuclei

The ratios of total histone to DNA for rat liver nuclei isolated by four methods as well as for calf liver nuclei isolated by one method were determined by obtaining the ratios of the total areas of the electrophoretic histone peaks for the liver nuclei to the corresponding total area given by a known amount of standard calf thymus histone. Ratios of total histone to DNA of approx. 2 for rat liver nuclei isolated at pH3.8 or 5.8 and for calf liver nuclei isolated at pH3.8 were confirmed twice by the above procedure and also by direct measurement, by the method of Lowry et al. (1951), of histone extracted in 0.2m-H2SO4. The histones of calf thymus, calf liver and rat liver were characterized by their amino acid compositions and by polyacrylamide-gel electrophoresis.

Studies of phosphorus metabolism by isolated nuclei. XII. Some fundamental properties of the incorporation of 32Pi into polyphosphate by rat liver nuclei

1984 ◽  
Vol 4 (11) ◽  
pp. 957-962 ◽  
Author(s):  
Ralph Penniall ◽  
James B. Griffin
Keyword(s):  
Rat Liver ◽  
Phosphorus Metabolism ◽  
Preliminary Estimate ◽  
Isolated Nuclei ◽  
Fundamental Properties ◽  
Rat Liver Nuclei ◽  
Minimal Rate ◽  
Liver Nuclei ◽  
Insoluble Product

Rat liver nuclei incubated in vitro catalyze a sustained incorporation of32Pi into polyphosphate. A preliminary estimate indicates a minimal rate of 10 moles of Pi incorporation into polyphosphates/h/mg protein. Polyphosphate is the predominant acid-insoluble product of nuclear phosphorylation; its formation is dependent on the presence of a divalent cation and is catalyzed by a system or systems as yet uncharacterized.

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