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.     

The nuclear dry weight of liver cells from patients with virus hepatitis and from controls.

Interferometric investigations were performed at liver cell nuclei isolated in 70 per cent glycerol. In 11 patients with virus hepatitis and seven patients without liver disease the nuclear dry weight of liver cells obtained by liver biopsy was determined by interferometry. The average nuclear dry weight of diploid liver cells from controls was 39.9 pg while an average value of 45.4 pg was found for patients with hepatitis. The corresponding nuclear volumes were 241 and 274mu3 respectively. The dry mass and volume of tetraploid nuclei was twice as big as that of diploid nuclei in both materials. The nuclear water content was neither significantly different between diploid and tetraploid nuclei nor significantly different between nuclei from controls and patients with hepatitis.     

Magnesium and Calcium in Isolated Cell Nuclei

The calcium and magnesium contents of thymus nuclei have been determined and the nuclear sites of attachment of these two elements have been studied. The nuclei used for these purposes were isolated in non-aqueous media and in sucrose solutions. Non-aqueous nuclei contain 0.024 per cent calcium and 0.115 per cent magnesium. Calcium and magnesium are held at different sites. The greater part of the magnesium is bound to DNA, probably to its phosphate groups. Evidence is presented that the magnesium atoms combined with the phosphate groups of DNA are also attached to mononucleotides. There is reason to believe that those DNA-phosphate groups to which magnesium is bound, less than 1/10th of the total, are metabolically active, while those to which histones are attached seem to be inactive.     

The isolation of cell nuclei in non-aqueous media

1. A modified Behrens procedure is described for the isolation of nuclei from avian erythrocytes and from the liver, kidney, thymus, pancreas, heart, and intestinal mucosa of the calf or horse. 2. The purity of these nuclei has been established by staining reactions, enzyme studies, and immunological tests for serum proteins. 3. Evidence is presented to show that a transport of cytoplasmic proteins into the nucleus does not occur during the isolation. 4. Nuclei prepared in non-aqueous media contain considerably more protein and a very different enzyme composition from that observed in nuclei prepared by "homogenization" techniques in dilute citric acid. 5. The suitability of nuclei prepared in organic media for the study of intracellular enzyme distribution is discussed.     

NMR-invisible ATP in rat heart and its change in ischemia

The subcellular compartmentalization of adenosine 5'-triphosphate (ATP) in isolated perfused rat heart and its relation to energy depletion in ischemia were examined by 31P nuclear magnetic resonance (31P-NMR) spectroscopy and chemical analyses. The signal intensities of the beta-phosphate of ATP and creatine phosphate in the 31P-NMR were standardized by the intracellular volume ratio measured with 23Na-NMR to determine the actual content of each. During aerobic perfusion the ATP content determined by NMR (13.7 +/- 2.2 mumol/g dry weight) was significantly lower than that found by chemical analysis (22.4 +/- 0.7 mumol/g dry weight), while the creatine phosphate contents determined by the two methods were the same. During ischemia at 33 degrees C, the signal of the beta-phosphate of ATP in the 31P-NMR spectrum decreased progressively, disappearing completely after 16 min. But at this time 5.7 +/- 1.7 mumol/g dry weight of myocardial ATP was still detected by chemical analysis. These results indicated that there were two different compartments of intracellular ATP in the heart, only one of which is detectable by 31P-NMR spectroscopy, and that during ischemia the ATP that is detectable, which seems to be the free ATP in the cytosol, decreased more rapidly than the ATP in the other compartment.     

Synchrony of Nuclear Replication in Individual Hyphae of Aspergillus nidulans

The synchrony of nuclear replication in individual, multinucleate hyphae of Aspergillus nidulans has been investigated. Samples were taken from cultures of germinating conidiospores, and the relative frequency of hyphae containing two to eight nuclei was determined. Because the conidiospores are mononucleate, complete synchrony will yield populations of hyphae containing only 2ⁿ nuclei, n being the number of doublings after germination. The appearance of hyphae with total numbers of nuclei other than 2ⁿ will indicate lack of synchrony. The relative frequency of hyphae not having 2ⁿ nuclei will depend on the degree of synchrony in the individual hyphae; numerical aspects of this relation are discussed. In two different strains, replication of the nuclei in any one hypha was highly synchronized when the dry weight doubling time was 1.4 to 1.8 hr. As the doubling time was made longer by changing the nitrogen or carbon source, synchrony was progressively lost. At the slowest growth rate tested, the interval between the division of the fastest and the slowest nucleus equaled 48% of the dry weight doubling time. The active replication of some nuclei in a hypha where other nuclei were resting suggested that nuclear duplication in this eukaryotic organism may be controlled by specific initiators.     

The distribution of nuclear and cytoplasmic proteins in the blastoderm of the loach, Misgurnus fossilis L

The concentration of dry substance (protein) and the dry weight of nuclei, cytoplasm and cells from different blastoderm regions at the early blastula and midgastrula stages were determined by interferentional microscopy. It was shown that at the early blastula stage the dry weight of cells in the basal layer is higher than that in the outer layer. Although the protein concentration in the basal layer cells appears to be somewhat higher, differences in their dry weight are due primarily to the big volume of cytoplasm of the basal layer cells. By the midgastrula stage, the total (nucleus + cytoplasm) protein concentration increases (by 17% in the basal layer cells and by 9% in the outer layer cells) due to the increase of nuclear protein concentration. At the same time dry weight of these cells markedly decreases due to the decrease of their volumes in the process of cell divisions. At the midgastrula stage the epiblast cells have the highest dry weight due to the highest protein concentration in the cytoplasm and the biggest cell volume. The results obtained are discussed with respect to the data on the pattern of accumulation of newly synthesized protein in nuclei and cytoplasm with special reference to the duration of individual cell cycle phases.     

Non-aqueous capillary electrophoresis of drugs: properties and application of selected solvents

The electrophoretic mobility of selected acidic and basic test solutes have been determined in non-aqueous media prepared by adding various combinations of ammonium acetate, sodium acetate, methane sulphonic acid and acetic acid to acetonitrile, propylene carbonate, methanol, formamide, N-methylformamide, N,N-dimethylformamide and dimethylsulphoxide, respectively. The apparent pH (pH*) of these non-aqueous media have been measured and it was found that pH* is an important factor for the separations in non-aqueous capillary electrophoresis. However, in some solvents the concentration of sodium acetate has a strong influence on the mobility despite very small changes in pH*. Due to the fact that a change in one parameter influences a number of other parameters it is very difficult to conduct systematic studies in non-aqueous media and to compare the migration of the species at fixed pH* values from one solvent to another. Thus pH* is only of value for comparison when used with a specific solvent or solvent mixture. The viscosity of the above-mentioned solvents were measured at various temperatures and means to adjust the viscosity of the non-aqueous media used for capillary electrophoresis are discussed and the separation of ibuprofen and its major metabolites in urine is used as an example.     

Engineering enzymes for non-aqueous solvents.

Enzymes may be redesigned to permit catalysis in non-aqueous solvents by engineering their amino acid sequences, thereby altering their physical and chemical properties to suit the new solvent environment. The interactions that contribute to protein stability in non-aqueous solvents are discussed in the context of attempting to identify possible approaches to constructing enzymes which exhibit enhanced stability in non-aqueous media. These approaches are illustrated by several examples where protein engineering has resulted in enzymes that are better suited for catalysis in organic solvents.     

The isolation of wheat germ nuclei and some aspects of their glycolytic metabolism

1. A procedure for isolating nuclei of the wheat germ in non-aqueous media has been described. 2. Such nuclei were shown to constitute about 50 per cent of the protoplasmic mass and to have a ribonucleic acid content of an order equivalent to that of the cytoplasm. 3. Studies of the distribution of the enzymes—aldolase, phosphoglyceraldehyde dehydrogenase, enolase, and pyruvate kinase—have revealed that the nuclei are the most vigorous sites of glycolytic activity. 4. Analysis of the DPN content of the nuclei in calf tissues—liver, pancreas, and heart—pointed to the probability that glycolytic activity is a characteristic common to many nuclei. 5. The significance of glycolytic activity to nuclear function has been discussed and some suggestive comparisons made between the two energy-yielding systems of glycolytic and oxidative phosphorylation.     

Differences in the condition of Norway lobsters (Nephrops norvegicus (L.)) from trawled and creeled fishing areas

The condition of trawled and creeled Norway lobsters, Nephrops norvegicus, was compared in individuals caught along the Swedish west coast. Female and male N. norvegicus were collected from trawled and creeled areas in the spring and autumn. Their nutritional state was determined by analysing individuals for haemocyanin concentration, the dry weight/wet weight relationship in muscle and hepatopancreas and the percentage dry weight of muscle and hepatopancreas of total animal dry weight. All parameters were measured on an individual basis and checked for size dependence. Creeled individuals were generally found to be in better condition than individuals from trawled areas, and animal condition increased from spring to autumn. Autumn males from creeled sites were overall in the best condition, having the highest mean haemocyanin concentration, muscle and hepatopancreas dry weight/wet weight relationship and percentage dry weight of muscle tissue. Claw symmetry (paired cutters) was suggested as an indicator of limb loss, with the highest occurrence found in females from trawled sites. Crusher absence had no effect on the animal's individual percentage dry weight of muscle, although the mean was lower in trawled females than in other groups. Resource limitation and physical stress are discussed as possible underlying factors affecting the observed differences.     

Changes in the nuclear polyamine content of chick erythrocytes during embryonic development.

The polyamine content of the circulating erythrocyte population in the embryonic chick was studied during its development. Total cellular polyamine content fell dramatically between 5 and 7 days of development, paralleling the decrease in metabolic activity exhibited by these cells. Nuclei were isolated from the erythrocytes by a non-aqueous technique, which not only eliminated the polyamine loss that occurred with aqueous isolation, but also prevented redistribution of the polyamines from the cytoplasm. Nuclear spermidine and spermine contents decreased markedly between 5 and 6 days of development from 31 to 10 pmol/microgram of DNA and from 33 to 18 pmol/microgram of DNA respectively. Thereafter the spermine content remained constant, but the spermidine content continued to decline. Good correlations between spermidine and RNA contents were observed in both cells and nuclei, and similarly between spermine and RNA contents in cells, but no such correlation was observed between spermine and RNA in nuclei.     

Xerotolerant mycobiota from high altitude Anapurna soil, Nepal

Xerophilic and xerotolerant microfungi were isolated from soil samples collected in Anapurna Mountains, Nepal, at altitudes from 3000 to 5400 m. The total numbers and proportions of xerotolerant and psychrophilic strains in comparison with mesophilic mycobiota were determined by using different enumeration, selective media and four isolation methods. The most extreme xerophilic fungi were taxonomically identified as belonging to the genera Eurotium and Aspergillus. The low water activity of the soil due to dry climate and frequent binding of water in ice crystals favors a high proportion of xerotolerant fungal species. The correlation between xerotolerant and psychrophilic fungi was observed.     

The hair melanosome: another tissue reservoir of zinc.

Melanosomes were isolated from human and dog hairs and their Zn content was determined. The mean Zn concentrations, in mug Zn/g dry weight, were 687 (men's melanosomes), 641 (women's melanosomes) and 691 (dog melanosomes). These values rank melanosomes from pigmented keratinous structures among the structural elements with the highest Zn content. An interpretation of the possible causes of this accumulation was submitted. By means of the conversion factor between hair length and weight (determined at 0.004), it was estimated that daily Zn lossess via hair vary in the region of 19 mug. In detailed studies of Zn metabolism, excretion via pigmented keratinous structures ought not to be neglected.     

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.     

Relation between the nuclei and the nucleoli during cell differentiation in roots of Vicia faba volumetric and cytochemical analysis

Summary The behaviour of the nuclei and the nucleoli of roots of Vicia faba during cell differentiation was studied quantitatively. The relations between these cell constituents and the polyploidy was analysed. The study was made on isolated nuclei and nucleoli and on plastic sections. A method for the isolation of nuclei and nucleoli of secondary roots fixed in formol was modified and another developed for material fixed in ethanol/acetic acid mixture. The volumetric investigation showed that the nuclear volume increases while the nucleolar decreases during cell differentiation. The mean number of nucleoli decreases. In Vicia faba there is no relation between the ploidy and the volume of nuclei and nucleoli; the protein synthesis rate has an influence on the size of these organelles. Quantitative investigation has shown the proportionality of dry weight, DNA, total protein, histone, protein-bound lysine and arginine content of the nuclei and their ploidy. The same experiments made on nucleoli showed linear relation between their content and volume. The concentration of analysed substances in constant in nucleoli.     

Isoenzymes of carbonic anhydrase of an euryhaline fish. Variations related to the osmoregulation]

A chemical study of carbonic anhydrase (EC 4.2.1.1) from the red blood cells and the gills of an euryhaline fish (Anguilla anguilla) is presented. Animals adapted to fresh water were compared to those adapted to sea water. The physiochemical constants of the various molecular formsisolated by chromatography and isoelectric focusing were determined; isoelectric pH, molecular weight, and the Km and V/E of the enzyme dehydration activity were compared. In both red cells and gills of fish adapted in either media various forms were isolated, characterized by different enzymatic kinetics (high- and low- activity forms) but having the same molecular weight (27 250). Some isoenzymes isolated from the gills differed significantly from those isolated from the red cells. Adaptation to fresh water or sea water is accompanied by modifications in the distribution of the isoenzymes in both red cells and gills: adaptation to sea water is characterized by a shift of the molecular forms towards an isoelectric pH higher than pH equals 6. The role of these enzymes is discussed under both a physiological and biochemical point of view in relation to the electrolyte exchange across fish gill. The origin of the different molecular forms of the carbonic anhydrase is discussed in relation to the prevailing theories on this subject.     

Determination of the sodium,potassium and chloride ion concentrations in the chloroplasts of the halophyteSuaeda maritima by non-aqueous cell fractionation

Summary Leaf material from the halophyteSuaeda maritima L. Dum. grown under both saline and non-saline conditions was fractionated under non-aqueous conditions in order to determine the ion content of various subcellular compartments. Fractions containing cell walls, nuclei and chloroplasts were successfully prepared and contents of DNA, chlorophyll, protein and Na⁺, K⁺, and Cl^– determined. The cell wall fraction was not apparently heavily contaminated by the other fractions and had a low ion content although the nuclear fraction was contaminated by other organelles. The ion contents of chloroplasts were determined and the results discussed in relation to earlier microscopical data.     

Production of prostacyclin, 6-keto-PGF1 alpha and thromboxane B2 by incubated samples of umbilical vessels: a comparison of methods for expressing the results on dry weight, wet weight, protein or DNA bases

The production of PGI2 (determined by bioassay), and of 6-keto-PGF1 alpha and TXB2 (determined by radioimmunoassay) by samples of human umbilical vessels have been measured. The results have been calculated on four bases: dry weight, wet weight, protein and DNA. There was a higher production of PGI2 and 6-keto-PGF1 alpha by umbilical veins than by umbilical arteries; no significant difference in TXB2 production was observed between umbilical veins and arteries. The ratio of 6-keto-PGF1 alpha: TXB2 production was about 100 for the samples of veins and about 40 for the samples of arteries. The best methods of expressing the results were on the bases of protein and DNA, the latter basis being marginally the best. The least satisfactory method for expressing the results was that based on dry weight. The physiological and practical implications of the results are discussed.