Soluble enzymes of nuclei isolated in sucrose and nonaqueous media; a comparative study

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.     

Occurrence of NI and NII type protein kinases in the nuclei from various tissues of the rat

Cyclic nucleotide-independent protein kinases that preferentially phosphorylated casein and phosvitin as substrate were surveyed in various tissue nuclei of the rat. Enzymes were extracted from the isolated nuclei of liver, kidney, spleen, brain, heart, or testis tissue with a buffer solution containing 0.4 m NaCl, and analyzed by DEAE-Sephadex, phosphocellulose, and Bio-Gel A-1.5m column chromatographies. The chromatographic study together with characterization of the enzymes demonstrated that all the tissues contained in their cell nuclei commonly two protein kinases, the NI and NII types, and that these were exclusively found as main nuclear casein kinases. NII enzyme activity was stimulated by polyamines and strongly inhibited by heparin. By contrast, the NI enzymes were little influenced by these compounds. We interpret the present results as suggesting that NI and NII type protein kinases may be found in the cell nuclei from many tissues of rat, and have distinct functions in the cell nuclei.     

Uptake and phosphorylation of phosphatidylinositol by rat liver nuclei. Role of phosphatidylinositol transfer protein

The incorporation of phosphatidyl[2-3H]inositol ([3H]PI) from vesicles or microsomal membranes into rat liver nuclei is greatly stimulated by phosphatidylinositol transfer protein (PI-TP). The nuclei are able to phosphorylate [3H]PI, with the production of phosphatidylinositol 4-phosphate (PIP). Recovery of tritiated inositol trisphosphate, inositol phosphate, glycerophosphoinositol and inositol, suggests that in isolated nuclei a large set of enzymes of the PI cycle is present, similar to the enzymes involved in the plasma membrane PI cycle. Incubation with [gamma-32P]ATP shows that isolated nuclei are able to phosphorylate endogenous PI to PIP and phosphatidylinositol 4,5-bisphosphate (PIP2). In the presence of exogenous PI and detergent the synthesis of PIP is increased, indicating that in nuclei the PI pool is suboptimal for the PI-kinase activity. The present study suggests that PI-TP may be involved in providing substrates for PI metabolism at the nuclear level.     

Occurrence of protein kinases NI and NII in human and porcine thyroids

Cyclic nucleotide-independent protein kinases that preferentially phosphorylated casein and phosvitin as substrate were detected in the nuclei of human and porcine thyroid tissues, and compared with those from rat liver. Enzymes were extracted from the isolated nuclei with a buffer solution containing 0.4 M NaCl, and analyzed by DEAE-Sephadex and phosphocellulose column chromatographies. The chromatographies, together with the characterization of the enzymes, demonstrated that human and porcine thyroid tissues contained two major casein kinases in the cell nuclei, the properties of which revealed that they are to be identified as protein kinases NI and NII.     

Effect of irradiation and endogenous nucleases on rat liver chromatin

These assessment of the consequences of irradiation on chromatin is complicated by endogenous nucleases. Isolation and prolonged storage of rat liver nuclei in buffers containing divalent metal ions activates these enzymes and promotes the degradation of chromatin. Irradiation of rat liver nuclei to dose levels of 20,000 rad under conditions in which endogenous nucleases are inhibited and analysis of the irradiated chromatin by sucrose density gradient centrifugation gave no evidence for monosomes or oligosomes. When chromatin from irradiated nuclei was digested with micrococcal nuclease, the levels of monosomes and oligosomes were identical to those of micrococcal nuclease, the levels of monosomes and oligosomes are identical to suggest that irradiation results in neither a direct fragmentation of linkers nor the sensitization of linkers for subsequent cleavage by micrococcal nuclease. Histones isolated from monosomes of irradiated and unirradiated nuclei were intact, showing no fragmentation or loss of residues, as judged by sodium dodecyl sulfate-polyacrylamide electrophoresis.     

ISOLATION AND PROPERTIES OF LIVER CELL NUCLEOLI

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.     

Creatine kinase from muscle cell nuclei]

It has been firstly demonstrated that rat heart and skeletal muscle nuclei contain creatine dinase, one of the most important enzymes of energy metabolism. The nuclei isolated in concentrated sucrose were practically free from cytoplasm and mitochondrial fragments. Electrophoresis in acetyl cellulose revealed that the nuclear extracts from rat heart and skeletal muscles contain only one isoenzyme of creatine kinase similar in mobility to the mitochondrial isoenzyme. The magnitude of Km values for creatine kinase from the nuclei of both tissues was determined. It was shown histochemically that creatine kinase is localized inside the nuclei, predominantly in the sites of chromatin location. A possible role of the enzyme in nuclear metabolism is discussed.     

Isolation and characterization of the nuclei fromSaccharomyces cerevisiae

We isolated highly intact nuclei from cells of a haploid strain ofSaccharomyces cerevisiae. The isolated nuclei were spherical, maintained a double membrane with typical nuclear pores, and were free from mitochondrial-, vacuolar-, and cytoplasmic-marker enzymes. The nuclei could synthesize both DNA and RNA and could phosphorylate nuclear proteins in vitro. These biochemical activities were greatly affected by the osmotic treatment of the nuclei.     

Hepatic cytochrome P-448 and epoxide hydrase: enzymes of nuclear origin are immunochemically identical with those of microsomal origin.

Nuclear and microsomal sources of hepatic cytochrome P-448 and epoxide hydrase were compared using antibodies made against the pure antigens isolated from rat liver microsomes. Both antigens were easily detected in detergent-solubilized nuclei and microsomes from rats using the Ouchterlony double-diffusion technique. Epoxide hydrase from either whole nuclei or nuclear envelope was immunochemically identical with the enzyme isolated from microsomes. Similarly, in rats pretreated with 3-methylcholanthrene, the cytochrome P-448 of nuclear origin was immunochemically indistinguishable from the enzyme derived from microsomes. These results establish the immunochemical identity of these hepatic nuclear and microsomal enzymes and provide a firm basis for applying the knowledge gained with the microsomal system of metabolism to the nuclear system.     

Poly(A) polymerases of rat liver nuclei. Purification and specificity.

Two poly(A) polymerases were isolated from rat liver nuclei and purified more than one thousand times by ion exchange chromatography on DEAE-Sephadex and phosphocellulose columns as well as affinity chromatography on a chromosomal RNA-Sepharose column. One of the two enzymes is bound to chromatin and uses as primer chromosomal RNA, while the second one is localized in the nucleoplasm and uses as primer poly(A) and hnRNA isolated from chromatin. The two enzymes seem to participate in the polyadenylation of chromosomal RNA in vitro, by a coupled mechanism. According to this mechanism, the chromatin bound enzyme adds 120-130 adenosine nucleotides to chromosomal RNA and consequently the nucleoplasmic enzyme completes the poly-adenylation by adding 80-90 more AMP units to the polyadenylated end of chromosomal RNA.     

Free amino acids and nucleic acid content of cell nuclei isolated by a modification of Behrens' technique

1. Nuclei were prepared from frozen rat liver by a modification of the technique of Behrens, and were studied with regard to the content of free amino acids and nucleic acid. 2. Under rigorously controlled conditions, preparations of nuclei are obtained by the Behrens' method which form a gel in the presence of 5 or 10 per cent NaCl or of water plus a small amount of dilute alkali; whereas when conditions are less rigorously controlled, nuclei are obtained which form no such gel. The property of forming gels with alkali is probably characteristic of all cell nuclei which have not undergone autolysis. 3. Nuclei prepared by the Behrens' technique contain the enzymes arginase, catalase, and esterase in very appreciable concentrations. 4. The free amino acids of the isolated cell nuclei, as well as of other liver cell fractions, have been investigated using the technique of paper chromatography. 5. The chromatographic patterns of the free amino acids of whole cells, ground cytoplasm, and isolated cell nuclei were very similar or identical. A feature of interest in these chromatograms was the faintness or absence of the spots due to a number of the essential amino acids, as compared to the intensities of the spots due to glycine, alanine, and glutamic acid. Glutathione was present in the isolated nuclei as well as in the whole cells. 6. Chromatograms made from hydrolysates of nuclei showed high concentrations of the essential amino acids and were similar to chromatograms of hydrolysates of typical proteins.     

Immunofluorescence revealed the presence of NHE-1 in the nuclear membranes of rat cardiomyocytes and isolated nuclei of human, rabbit, and rat aortic and liver tissues

Using immunofluorescence and 3-dimensional confocal microscopy techniques, the present study was designed to verify if NHE-1 is present at the level of the nuclear membrane in cells that are known to express this type of exchanger. Nuclei were isolated from aortic tissues of adult human, rabbit, and rats, as well as from liver tissues of human fetus, and adult rabbit and rat. In addition, cultured ventricular cardiomyocytes were isolated from 2-week-old rat. Our results showed the presence of NHE-1 in isolated nuclei of aortic vascular smooth muscle and liver of human, rabbit, and rat. NHE-1 seems to be distributed throughout the isolated nucleus and more particularly at the level of the nuclear membranes. The relative fluorescence density of NHE-1 was significantly higher (p < 0.05) in isolated liver nuclei of human, when compared with those of rabbit and rat. However, in isolated nuclei of aortic vascular smooth muscle, the relative fluorescence density of NHE-1 was significantly (p < 0.001) higher in the rabbit when compared with human and rat. In cultured rat ventricular cardiomyocytes, NHE-1 fluorescent labeling could be easily seen throughout the cell, including the nucleus, and more particularly at both the sarcolemma and the nuclear membranes. In rat cardiomyocytes, the relative fluorescence density of NHE-1 of the sarcolemma membrane, including the cytosol, was significantly lower than that of the whole nucleus (including the nuclear envelope membranes). In conclusion, our results showed that NHE-1 is present at the nuclear membranes and in the nucleoplasm and its distribution and density may depend on cell type and species used. These results suggest that nuclear membranes' NHE-1 may play a role in the modulation of intranuclear pH.     

A cell line with decreased sensitivity to the methyl mercury-induced stimulation of alpha-amanitin sensitive RNA synthesis in isolated nuclei

1. In nuclei isolated from cells of the B50 rat neuroblastoma line the stimulatory effect of methyl mercury on alpha-amanitin-sensitive RNA synthesis is very much reduced compared to the stimulatory effect in HeLa nuclei (see: Frenkel G. D. and Randles K. (1982) Specific stimulation of alpha-amanitin-sensitive RNA synthesis in isolated HeLa nuclei by methyl mercury. J. biol. Chem. 257, 6275-6279). 2. The stimulatory effect of another mercury compound, p-hydroxymercuribenzoate, was also much less pronounced in the B50 nuclei. 3. Similar results were obtained with nuclei isolated from B50 cells which had been induced to differentiate by exposure to dibutaryl cyclic AMP. 4. Nuclei isolated from cells of another rat neuroblastoma line (B35), and nuclei from cells of a human neuroblastoma line both exhibited levels of stimulation similar to that of HeLa nuclei. 5. The B50 and HeLa cells were also compared as to their sensitivity to other effects of methyl mercury.     

大白鼠DNA拓扑异构酶Ⅰ生物学性质的研究

对大白鼠组织作DNA拓扑弄构酶Ⅰ(拓扑酶Ⅰ)活力测定,见酶活力出现在胚胎早期,在胚胎发育过程及出生后不同年龄期,酶活力基本稳定;几种成年大鼠组织的酶活力彼此无显著差异;肝细胞再生及癌变,酶活力亦无显著变化。     

Discrete subcellular localization of phosphoinositidase C beta, gamma and delta in PC12 rat pheochromocytoma cells.

Phosphoinositidase C activity was revealed in nuclei isolated from PC12 rat pheochromocytoma cells incubated with tritiated phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Phosphoinositide breakdown was found to be optimal at neutral pH and Ca++ concentrations ranging from endogenous levels to millimolar values. To characterize the enzymes involved, three monoclonal antibodies directed against the beta, gamma and delta phosphoinositidase C isoforms were employed. A combination of Western blot immunochemical analysis on cytoplasmic and nuclear fractions and of in situ immunocytochemistry on intact cells and isolated nuclei indicated that phosphoinositidase C gamma, though predominantly cytoplasmic, was present in both cell compartments. On the contrary, phosphoinositidase C beta was exclusively localized in the nucleus, whereas phosphoinositidase C delta was restricted to the cytoplasm. These data suggest that inositol lipid breakdown is controlled by different phosphoinositidase C isozymes in the various cell compartments, and support the notion that a separate phosphoinositide signalling system is located in the nucleus.     

Nuclear deoxyribonucleic acid polymerases of liver.

Hepatic nuclei that are isolated in aquenous solutions of low ionic strength or glycerol contain all or nearly all the nonmitochondrial DNA polymerase activity of the cell. The presence of polymerase activity in the cytoplasm is due to extraction of nuclear enzymes by buffer and inorganic salts. Even with low ionic strength solutions, some leaching of nuclear enzymes occurs if the concentration of liver in the homogenizing medium is greater than 10%. As defined by sucrose gradient analysis, the normal adult rat liver nucleus contains mainly or entirely a single species of DNA polymerase (3.2 S) whereas the regenerating nucleus after 70% hepatectomy has an additional enzyme (7.1 S). The total activity of regenerating nuclei is about twice the normal value. The increase resides in the 7.1 S activity. The 7.1 S DNA polymerase had been purified partially from regenerating liver nuclei (isolated in low ionic strength solutions) and cytosol (prepared under conditions of nuclear enzyme extraction). The properties of the activity from the two sources are indistinguishable. A mixture of albumin and spermidine enhances by several-fold the activities of the 3.2 S and 7.1 S DNA polymerases. In the presence of spermidine, but not in its absence, the activity of the 7.1 S DNA polymerase is strictly proportional to the amount of the enzyme preparation.     

Estrogen- and progestin-receptor complexes and their interactions with rat liver cell nuclei in ontogenesis

Steroid-receptor complexes (SRC) of estrogen and progestin were isolated from rat liver and purified 1500-2000-fold. The SRC within the composition of cytosol and purified 2000-fold were characterized by gel filtration of Sephadex G-100 and by DEAE-cellulose chromatography. The purified SRC from rat liver were bound to isolated liver cell nuclei of rats of various age (1.5, 6, 12 and 24 month-old). The maximal binding of progestin and estrogen SRC from rat liver was observed in homologous nuclei of 1.5-month-old animals. The binding of SRC by the nuclei decreased progressively with age, reaching its minimum in 24-month-old rats. The observed differences in the SRC binding by cell nuclei of experimental animals may be the cause of functional changes at various stages of ontogenesis.     

The glutathione status of rat kidney nuclei following administration of buthionine sulfoximine

Rat kidney nuclei were isolated by techniques designed to limit the loss of small water-soluble metabolites. Lyophilized rat kidney powder was disrupted with a Tekmar Tissumizer, and nuclei were purified by discontinuous gradient centrifugation through a glycerol-metrizamide solution at 4 degrees C. Purified nuclei exhibited similar DNA to protein ratios as reported for other rat nuclei isolated by nonaqueous isolation techniques. Results suggest that in control animals the concentration of glutathione in the kidney nucleus is similar to that in the cytoplasm. However, following treatment with buthionine sulfoximine, rat kidney nuclear glutathione levels were less than the corresponding cytoplasmic glutathione levels.     

Proteolytic digestion of histones indicates that the nucleosomes of nuclei and of sheared chromatin are similar

Calf thymus nuclei were treated with trypsin, chymotrypsin or Pronase, and the rate of digestion of the various histone fractions was determined. The results differed from those obtained by digestion of DNA-free histones with the same set of enzymes but were identical to those obtained by digestion of calf thymus chromatin. Because these enzymes have such different specificities, the results of these digestions indicate that the histone fractions have similar locations in the chromosomal substructures of nuclei and chromatin, $\text{i.e.}$ that the structure of the nucleosomes which exist within nuclei is not changed markedly when chromatin is isolated from nuclei by a method which involves shearing.