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 Corticotropin-(1–24)-tetracosapeptide affects protein phosphorylation and polyphosphoinositide metabolism in rat brain

1981 ◽  
Vol 194 (1) ◽  
pp. 283-291 ◽  
Author(s):  
J Jolles ◽  
H Zwiers ◽  
A Dekker ◽  
K W A Wirtz ◽  
W H Gispen
Keyword(s):  
Phosphatidic Acid ◽  
Protein Phosphorylation ◽  
Subcellular Fraction ◽  
Membrane Phospholipids ◽  
Cytosol Fraction ◽  
Phosphatidylinositol Bisphosphate ◽  
Phosphorylation Of Proteins ◽  
Synaptosomal Fraction ◽  
Phosphatidylinositol Phosphate ◽  
Polyphosphoinositide Metabolism

1. Effects of corticotropin-(1–24)-tetracosapeptide on the endogenous phosphorylation of proteins and lipids were studied in a membrane/cytosol fraction prepared from a lysed crude mitochondrial/synaptosomal fraction. 2. The labelling of proteins and lipids was monitored by incubation of the subcellular fraction for 10s with [gamma-32P]ATP. 3. The phosphorylation of proteins was dose-dependently inhibited by the peptide (40% of control incubations at 100 microM-corticotropin). 4. Of the membrane phospholipids only phosphatidylinositol phosphate, phosphatidylinositol bisphosphate and phosphatidic acid became labelled. Corticotropin dose-dependently increased the formation of phosphatidylinositol bisphosphate and inhibited the production of phosphatidic acid (470% and 50% respectively of control incubations, at 100 microM of the peptide) and had no effect on phosphatidylinositol phosphate. 5. Phosphatase activity was observed to act on phosphatidylinositol bisphosphate, phosphatidylinositol phosphate and phosphoprotein but not on phosphatidic acid. 6. Corticotropin interacted with the kinases rather than with the phosphatases. 7. The formation of phosphatidylinositol bisphosphate and phosphatidic acid was maximal at 1–10mM-Mg2+ in the absence of Ca2+, and the production of phosphatidylinositol phosphate was maximal at 30mM-Mg2+. 8. The basal value of lipid phosphorylation decreased with increasing Ca2+ concentration. 9. Ca2+ abolished the effect of corticotropin on phosphatidylinositol bisphosphate formation (470%, 190% and 100% of control incubations at respectively 0, 0.1 and 1 mM-Ca2+). 10. The data provide evidence that the effects of corticotropin on protein phosphorylation and on polyphosphoinositide metabolism in brain membranes are related.

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.

Stimulation of phospholipase D activity and phosphatidic acid production by norepinephrine in rat aorta

1993 ◽  
Vol 264 (3) ◽  
pp. C609-C616 ◽  
Author(s):  
A. W. Jones ◽  
S. D. Shukla ◽  
B. B. Geisbuhler
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Time Course ◽  
Rat Aorta ◽  
Functional Responses ◽  
Phosphatidylinositol Bisphosphate ◽  
Phosphatidylinositol Phosphate ◽  
Transphosphatidylation Reaction ◽  
Hydrolysis Of ◽  
Stimulation Of

We sought to relate norepinephrine (NE) stimulation of phosphatidic acid (PA) production to functional responses of rat aorta and pathways for PA production. The time course for changes in PA was closely related to Ca-dependent tonic responses in 42K efflux and contraction. NE (30 microM for 1 min) increased PA and reduced phosphatidylcholine (PC) and phosphatidylinositol (PI) based on Pi analyses and 32P labeling of phospholipids. The 32P-to-Pi ratio in PA (0.8 +/- 0.2, n = 13) was similar to PC (0.8 +/- 0.1, n = 14) but was significantly lower (P < 0.001) than PI (4.6 +/- 0.5, n = 14). The 32P-to-Pi ratio in PA was also lower (P < 0.02) than phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. NE also increased [3H]PA twofold (P < 0.05) when PC was selectively labeled with [3H]myristic acid. These observations are more consistent with PA being formed from the hydrolysis of PC by phospholipase D (PLD) than by the phosphorylation of diacylglycerol produced by the action of phospholipase C. PLD was assayed by the formation of phosphatidylethanol (PEt) via a transphosphatidylation reaction with ethanol (half-maximal stimulation at 0.4-0.5% vol/vol). The time course for PLD stimulation by NE was similar to PA, with significant increases (P < 0.002) during 10 s to 30 min exposure. Once formed, PEt was degraded slowly, with a half time > 3 h. It is concluded that NE stimulates PLD in rat aorta, which forms a significant amount of PA from the hydrolysis of PC.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

The Triton X-100 and High Salt Resistant Residue of Saccharomyces cerevisiae Nuclear Membranes

1982 ◽  
Vol 37 (10) ◽  
pp. 916-920 ◽  
Author(s):  
Karlheinz Mann ◽  
Dieter Mecke
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Membrane ◽  
Initial Treatment ◽  
High Salt ◽  
Isolated Nuclei ◽  
Nuclear Membranes ◽  
Higher Eukaryotes ◽  
Triton X

Abstract Saccharomyces cerevisiae nuclear membranes were prepared from isolated nuclei by digesting chromatin with deoxyribonuclease after an initial treatment of nuclei with very diluted buffers. When the nuclear membranes were treated with 5% Triton X-100 and 1 ᴍ NaCl an insoluble fibrous net was obtained which consisted mainly of protein with Mr values of 85000, 48000, 45000, 39000 and 31000. Lamins, a set of proteins with Mr = 65000-75000, which were shown to be the major proteins of the insoluble nuclear membrane residue of higher eukaryotes, were not found.

Thyrotrophin-releasing hormone stimulates polyphosphoinositide metabolism in the frog neurointermediate lobe

1990 ◽  
Vol 5 (2) ◽  
pp. 129-136 ◽  
Author(s):  
L. Desrues ◽  
M. C. Tonon ◽  
H. Vaudry
Keyword(s):  
Pars Intermedia ◽  
Rapid Loss ◽  
Isotopic Equilibrium ◽  
Thyrotrophin Releasing Hormone ◽  
Phosphatidylinositol Bisphosphate ◽  
Maximal Stimulation ◽  
Rat Pituitary ◽  
Rapid Stimulation ◽  
Stimulation Of ◽  
Polyphosphoinositide Metabolism

ABSTRACT Previous studies have demonstrated that TRH is a potent stimulator of α-MSH secretion from frog pituitary melanotrophs. In order to determine the intracellular events responsible for TRH-evoked α-MSH release, we have investigated the effect of TRH on polyphosphoinositide breakdown in frog pars intermedia. Neurointermediate lobes were labelled to isotopic equilibrium with myo-[3H]inositol. TRH stimulated the rate of incorporation of [3H]inositol into the phospholipid fraction. The effect of TRH was concentration-dependent; half-maximal stimulation of α-MSH release and inositol incorporation occurred at 12 and 28 nmol TRH/1 respectively. In prelabelled neurointermediate lobes, lithium (10 mmol/l) enhanced the radioactivity in inositol monophosphate, bisphosphate (IP2) and trisphosphate (IP3). LiCl (10 mmol/l) induced a 38% inhibition of α-MSH release from perifused neurointermediate lobes but did not impair TRH-induced α-MSH secretion. In the presence of LiCl, TRH (1 μmol/l) induced a transient increase of the radioactivity in IP3, which was evident by 30 s and maximal by 1 min (+ 100%). TRH treatment also increased the radioactivity in IP2, which reached a plateau after 5 min (+ 100%). The increase in radioactivity in IP3 induced by TRH was closely paralleled by a rapid loss of [3H]phosphatidylinositol bisphosphate (PIP2), which was maximal by 1 min (−70%). These results indicate that, in frog pars intermedia, TRH-evoked α-MSH secretion is coupled to breakdown of PIP2. The data suggest that, in amphibian melanotrophs, as previously shown in GH3 tumour cells and in rat pituitary mammotrophs, TRH causes rapid stimulation of polyphosphoinositide-hydrolysing phospholipase C.

scholarly journals ISOLATION AND PROPERTIES OF LIVER CELL NUCLEOLI

1956 ◽  
Vol 2 (2) ◽  
pp. 127-145 ◽  
Author(s):  
K. J. Monty ◽  
M. Litt ◽  
E. R. M. Kay ◽  
A. L. Dounce
Keyword(s):  
Liver Cell ◽  
Cell Type ◽  
Isolated Nuclei ◽  
Dna And Rna ◽  
Detailed Method ◽  
Liver Nuclei ◽  
Specific Activities ◽  
New Hypothesis ◽  
Isolated Liver ◽  
Isolated Rat

1. The significance of the term nucleolus has been discussed. 2. A detailed method for the isolation of nucleoli from already isolated rat or cat liver nuclei has been presented. 3. The presence of DNA in isolated liver cell nucleoli has been indicated by histochemical methods. 4. The percentages of DNA and RNA in the isolated nucleoli have been determined by chemical analysis. 5. The specific activities of aldolase, arginase, and catalase have been determined for two subnuclear fractions and for the isolated nucleoli of rat and cat liver, and the relative amounts of these enzymes in the same subnuclear fractions and nucleoli of rat liver have been measured. 6. The significance of the above findings has been discussed and consideration has been given to what types of isolated nuclei might best serve as starting material for the isolation of nucleoli. 7. A new hypothesis has been presented that nucleoli of the liver cell type may function primarily in furnishing (directly or indirectly) templates for the synthesis of the particular enzymes that must govern the chemistry of mitosis.

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