A COMPARISON OF NUCLEAR DRY WEIGHTS DETERMINED BY CHEMICAL AND BY INTERFEROMETRIC METHODS

1. The nuclei of cells from the thymus of the calf were isolated by three different techniques; the citric acid, the sucrose-calcium chloride, and the non-aqueous. 2. The mean dry weights of the nuclei were determined by chemical methods and by microscopic interferometry. There was a close correlation between the results from the interferometric and chemical methods. 3. The range of values about that mean was determined in each sample: the nuclei isolated in aqueous media contained approximately 45 per cent less material than those isolated in non-aqueous media. 4. The variations in dry weight with varying nuclear type are discussed. 5. The possible relationship between DNA content and dry weight is discussed.     

Role of alkaline endonucleases in the release of soluble chromatin from thymus, spleen and liver nuclei of normal and irradiated mice.

Thymus, spleen and liver nuclei released a large fraction of soluble chromatin in vitro when incubation was carried out in sucrose media containing low concentrations of CaCl2 and/or MgCl2. A significant fraction of deoxyribopolynucleotides (DPN) was also extracted from nuclei. After 30 min of incubation at 37 degrees C, the maximum release of soluble chromatin was observed near a pH of 8, which corresponds to the optimum pH of the alkaline endonuclease activity from thymus, spleen and liver. The soluble chromatin and DPN were precipitated by increasing the bivalent ion concentration of the medium. The protein/DNA ratio and the molecular weight of DNA suggest that the soluble chromatin and DPN represent nucleosome-like particles. The release of soluble chromatin in the first 4 hours of incubation was significantly increased if the nuclear fraction was isolated from the thymus and spleen of whole-body irradiated mice (1000 rad). This effect was absent in the liver nuclei.     

High total histone/deoxyribonucleic acid ratios for rat 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-H(2)SO(4). The histones of calf thymus, calf liver and rat liver were characterized by their amino acid compositions and by polyacrylamide-gel electrophoresis.     

Cytosolic enzyme leakage from isolated smooth muscle preparations

Several isolated smooth muscle preparations were shown to release cytosolic enzymes including adenosine deaminase, lactate dehydrogenase, and nucleoside phosphorylase under experimental conditions resembling those used in contractility or drug uptake studies. This enzyme leakage indicates significant general cell damage and suggests that experiments dealing with drug metabolism, binding, and uptake in isolated tissues must be interpreted with caution.     

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.     

Adenosine, deoxyadenosine, and deoxyguanosine induce DNA cleavage in mouse thymocytes

When thymocytes were cultured with adenosine, deoxyadenosine, or deoxyguanosine at 1 mM for 24 h, DNA cleavage at internucleosomal sites with multiples of approximately 180 bp was induced, followed by lactate dehydrogenase release into the medium. In the presence of coformycin, an adenosine deaminase inhibitor, or formycin B, a purine nucleoside phosphorylase inhibitor, DNA cleavage was induced by these nucleosides at concentrations of less than 50 microM. Other purine and pyrimidine ribo- and deoxyribonucleosides did not induce DNA cleavage or LDH release. Because thymocyte nuclei contain a Ca2+,Mg2+-dependent endonuclease, which preferentially cuts DNA in its linker regions, DNA fragmentation induced by the three purine nucleosides was suggested to occur through increased activity of the endonuclease. The DNA cleavage induced by the nucleosides required protein phosphorylation and synthesis, inasmuch as it was inhibited by an inhibitor of protein kinases, H-7, and by an inhibitor of protein synthesis, cycloheximide. The inhibition of DNA cleavage was accompanied by a reduction in lactate dehydrogenase release, suggesting a causal relationship between DNA cleavage and cell death. The DNA cleavage and subsequent cell lysis might be related to the selective thymocyte deletion observed in patients with adenosine deaminase or purine nucleoside phosphorylase deficiency.     

Purine catabolism in rat brain (author's transl)

In soluble rat brain fraction, the specific activities of purine nucleoside phosphorylase, guanine deaminase, 5'Nucleotidase and adenosine deaminase, decrease in their mentioned order. A kinetic parameter comparison between these enzymes shows that 5'Nucleotidase with AMP has the lowest KM and the greatest Vmax values, while purine nucleoside phosphorylase has its lowest KM and its greatest Vmax values with guanosine and with inosine, respectively. The enzymes activity is not modified by the metabolic intermediates differently from their own reaction products which behave as competitive inhibitors.     

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.     

Genetic studies of free-ranging macaques of Cayo Santiago. I. Description of the population and some nonpolymorphic red cell enzymes.

Phenotypes of eight red cell enzymes at nine genetic loci were determined in the semi-free-ranging population of rhesus macaques; Macaca mulatta, that inhabit Cayo Santiago. The following enzymes were examined electrophoretically: adenosine deaminase, glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, indophenol oxidase, lactate dehydrogenase, malate dehydrogenase, phosphoglucomutase-1, phosphoglumutase-2, and purine nucleoside phosphorylase. Hemolysates from at least 372 animals were analyzed, and no variants of the enzymes were observed with the exception of malate dehydrogenase. Three animals displaying a variant form of malate dehydrogenase were found.     

Induction and repression of enzymes involved in exogenous purine compound utilization of Bacillus cereus

5'-Nucleotidase, adenosine phosphorylase, adenosine deaminase and purine nucleoside phosphorylase, four enzymes involved in the utilization of exogenous compounds in Bacillus cereus, were measured in extracts of this organism grown in different conditions. It was found that adenosine deaminase is inducible by addition of adenine derivatives to the growth medium, and purine, nucleoside phosphorylase by metabolizable purine and pyrimidine ribonucleosides. Adenosine deaminase is repressed by inosine, while both enzymes are repressed by glucose. Evidence is presented that during growth of B. cereus in the presence of AMP, the concerted action of 5'-nucleotidase and adenosine phosphorylase, two constitutive enzymes, leads to formation of adenine, and thereby to induction of adenosine deaminase. The ionsine formed would then cause induction of the purine nucleoside phosphorylase and repression of the deaminase. Taken together with our previous findings showing that purine nucleoside phosphorylase of B. cereus acts as a translocase of the ribose moiety of inosine inside the cell (Mura, U., Sgarrella, F. and Ipata, P.L. (1978) J. Biol Chem. 253, 7905-7909), our results provide a clear picture of the molecular events leading to the utilization of the sugar moiety of exogenous AMP, adenosine and inosine as an energy source.     

Intranephron distribution of purine-metabolizing enzymes in rats

The activities of purine-metabolizing enzymes, 5'-nucleotidase, adenosine deaminase, and purine nucleoside phosphorylase in microdissected rat nephron segments were measured. The specific activity of 5'-nucleotidase was highest in the proximal tubules and the cortical collecting duct, but low in the glomerulus. In contrast, the highest activity of adenosine deaminase was found in the glomerulus. The distribution pattern of purine nucleoside phosphorylase was similar to that of adenosine deaminase. These results suggest that various nephron segments can form adenosine and that the glomerulus exhibits highest capacities to metabolize this nucleoside.     

Mutants of Escherichia coli Defective in Ribonucleoside and Deoxyribonucleoside Catabolism

From Escherichia coli B, mutants were prepared that lacked the enzymes adenosine deaminase, cytidine deaminase, and purine nucleoside phosphorylase. In each case, the mutant lacked enzyme activity for both ribonucleoside and deoxyribonucleoside. Mutants lacking purine nucleoside phosphorylase lost the capacity to cleave the nucleosides of adenine, guanine, and hypoxanthine.     

Induction and repression of enzymes involved in exogenous purine compound utilization in Bacillus cereus

5′-Nucleotidase, adenosine phosphorylase, adenosine deaminase and purine nucleoside phosphorylase, four enzymes involved in the utilization of exogenous purine compounds in Bacillus cereus, were measured in extracts of this organism grown in different conditions. It was found that adenosine deaminase is inducible by addition of adenine derivatives to the growth medium, and purine nucleoside phosphorylase by metabolizable purine and pyrimidine ribonucleosides. Adenosine deaminase is repressed by inosine, while both enzymes are repressed by glucose. Evidence is presented at during growth of B. cereus in the presence of AMP, the concerted action of 5′-nucleotidase and adenosine phosphorylase, two constitutive enzymes, leads to formation of adenine, and thereby to induction of adenosine deaminase. The ionsine formed would then cause induction of the purine nucleoside phosphorylase and repression of the deaminase. Taken together with our previous findings showing that purine nucleoside phosphorylase of B cereus acts as a translocase of the ribose moiety of ionsine inside the cell (Mura, U., Sgarrella, F. and Ipata, P.L. (1978) J. Biol. Chem. 253, 7905–7909), our results provide a clear picture of the molecular events leading to the utilization of the sugar moiety of exogenous AMP, adenosine and inosine as an energy source.     

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

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.     

Enzymatic synthesis of purine 2'-deoxyriboside and its properties as an inhibitor of adenosine deaminases from calf intestinal mucosa and Bacillus cereus

Enzymatic synthesis of purine 2'-deoxyriboside was obtained by reacting purine with excess 2-deoxy-alpha-D-ribose-1-phosphate in the presence of commercial bovine nucleoside phosphorylase; the product was isolated by semipreparative reverse phase HPLC with an overall 62% yield. Purine 2'-deoxyriboside was shown to behave as a competitive inhibitor of adenosine deaminase from calf intestinal mucosa and Bacillus cereus, with apparent Ki values of 4.5 and 8.5 microM, respectively.     

Interaction of the calf uterine estrogen receptor with acceptor sites in heterologous chicken target cell nuclei

Estrogen receptor (ER) from chicken liver and calf uterus were used to study the capacity and the characteristics of the receptor binding sites (acceptor sites) in chicken target cell nuclei. Binding studies were performed at a physiological salt concentration of 0.15 M KCl. Binding of liver ER to liver nuclei was temperature-dependent, showing a 9-fold increase between 0 and 28 degrees C. The maximal number of acceptor sites measured in this cell-free system (280 sites/nucleus) was considerably lower than measured in nuclei after in vivo administration of estrogen (820 sites/nucleus). Moreover incubation of nuclei with the liver ER preparation resulted in a substantial breakdown of nuclear DNA, making this ER less suitable for DNA binding studies. The temperature-activated calf uterine receptor bound to liver nuclei at 0 degrees C, at which temperature no DNA degradation was measured. To all chicken cell nuclei tested, the receptor bound with a high affinity (Kd = 0.4-1.0 nM). Nuclear binding displayed tissue specificity: oviduct greater than heart, liver greater than spleen greater than erythrocytes and was salt dependent. Calf uterine ER binding in liver nuclei ranged from 3000-6000 acceptor sites per nucleus when assayed under conditions of a constant protein or a constant DNA concentration. Nuclei isolated from estrogen-treated cockerels bound a 2-fold lower number of calf uterine ER complexes when compared to control nuclei. Incubation of nuclei with a fixed concentration of [3H]ER from liver and increasing concentrations of uterine non-radioactive-ER also resulted in a reduced binding of the liver receptor. Both types of experiments suggest that liver and uterine ER compete for a common nuclear acceptor site. Our data demonstrate that the ER from calf uterus is very useful as a probe to examine the nature of the acceptor sites in heterologous chicken target cell nuclei. The assay system functions at 0 degrees C, a temperature at which no DNA degradation occurs.     

Some enzymes of isolated nuclei

THE COMPOSITION OF ISOLATED NUCLEI AND CELL PREPARATIONS FROM TISSUES OF CALF, BEEF, HORSE, AND FOWL WAS STUDIED WITH RESPECT TO THE FOLLOWING COMPONENTS: 1. Liver and kidney arginase, catalase, and uricase; pancreatic lipase and amylase; cardiac muscle myoglobin; erythrocyte hemoglobin; intestinal alkaline phospharase. These are referred to as "special" components in view of their characteristically restricted distribution reflecting the differentiated nature of the tissues in question. 2. Esterase, beta-glucuronidase, alkaline and nucleotide phosphatases, adenosine deaminase, guanase, and nucleoside phosphorylase. These are enzymes of general distribution. The differences in nuclear composition noted with respect to the "special" components, together with the broad variability in nuclear activity found for enzymes of general distribution, led to the conclusion that nuclei are differentiated structures. The following distribution was observed: 1. "Special" components: Hemoglobin was found to be present in fowl and goose erythrocyte nuclei, but myoglobin was entirely absent from heart muscle nuclei; of the special enzymes listed, only catalase and arginase appeared to be concentrated in some of the nuclei. There was no significant nuclear concentration of lipase, amylase, uricase, or alkaline phosphatase. No simple relationship was found between the concentration of a special enzyme in a tissue and its activity in the corresponding nuclei. For example, arginase activity, which is high in mammalian liver and in fowl kidney, was found in liver, not kidney, nuclei. Similarly, catalase activity was demonstrated only in mammalian liver nuclei, although, in mammals, both liver and kidney are rich sources of this enzyme. 2. Enzymes of general distribution fell into three classes: (a) Those present in low concentrations, if at all, in the nuclei-alkaline phosphatase, the nucleotide phosphatases) and beta-glucuronidase. (b) Those present in nuclei in varying concentrations-esterase. (c) Those present in high proportions in most nuclei-adenosine deaminase, nucleoside phosphorylase, and guanase. The exceptionally low nuclear activity of intestinal mucosa with respect to these enzymes was discussed in relation to physiological considerations. The response of nuclei to changes in physiological state was demonstrated by experiments on starvation. The outstanding aspect of this response was a change in nuclear enzymatic activity opposing that observed in the cytoplasm. A comparison of fetal and adult mucosa cells led to the following tentative interpretation of the observed intracellular enzyme distribution: In cells tending to moribundity, as in those subjected to starvation, relative nuclear enzymatic activity falls. The occurrence of special enzymes in nuclei was considered in terms of differentiation, and the high nuclear concentration of the nucleoside-specific enzymes was interpreted in terms of general nuclear metabolic activity.     

METHODS FOR THE PURIFICATION OF THYMUS NUCLEI AND THEIR APPLICATION TO STUDIES OF NUCLEAR PROTEIN SYNTHESIS

Procedures are described for the purification of calf thymus nuclei using mild hypotonit shock to break intact cells, and layering techniques to remove cytoplasmic debris. Ficolc (a high polymer of sucrose) was dissolved in isotonic sucrose to give dense solutions suitable for gradient centrifugation. The method yields nuclei which can incorporate amino acids in vitro. Thymus nuclei isolated under isotonic conditions were incubated with C¹⁴-amino acids and later purified by centrifugation through dense sucrose solutions. The distribution of radioactivity in different nuclear proteins was measured and it was found that isotopic amino acids are actively incorporated into characteristically chromosomal proteins, such as the arginine-rich and lysine-rich histones. Protein synthesis in the nucleus is markedly inhibited by puromycin and by agents, such as 2,4-dinitrophenol, which inhibit ATP synthesis. The synthesis of histones is also inhibited by puromycin, but the uptake of several amino acids into the lysine-rich histone fraction seems less sensitive to puromycin inhibition than is uptake into the arginine-rich histones or other proteins of the nucleus. High resolution autoradiography using tritiated leucine and observing grain distribution over thin sections of isolated nuclei and whole cells shows that amino acid incorporation occurs within the nucleus and is not due to cytoplasmic contamination.     

Specificity of the histone lysine methyltransferases from rat brain chromatin

The histone lysine methyltransferases catalyze the transfer of methyl groups from S-adenosylmethionine to specific epsilon-N-lysine residues in the N-terminal regions of histones H3 and H4. These enzymes are located exclusively within the nucleus and are firmly bound to chromatin. The chromosomal bound enzymes do not methylate free or nonspecifically associated histones, while histones H3 and H4 within newly synthesized chromatin are methylated. These enzymes can be solubilized by limited digestion (10-16%) of chromosomal DNA from rapidly proliferating rat brain chromatin with micrococcal nuclease. Histone H3 lysine methyltransferase remained associated with a short DNA fragment throughout purification. Dissociation of the enzyme from the DNA fragment with DNAase digestion resulted in complete loss of enzyme activity; however, when this enzyme remained associated with DNA it was quite stable. Activity of the dissociated enzyme could not be restored upon the addition of sheared calf thymus or Escherichia coli DNA. Histone H3 lysine methyltransferase was found to methylate lysine residues in chromosomal bound or soluble histone H3, while H3 associated with mature nucleosomes was not methylated. The histone H4 lysine methyltransferase which was detectable in the crude nuclease digest was extremely labile, losing all activity upon further purification. We isolated a methyltransferase by DEAE-cellulose chromatography, which would transfer methyl groups to arginine residues in soluble histone H4. However, this enzyme would not methylate nucleosomal or chromosomal bound histone H4, nor were methylated arginine nucleosomal or chromosomal bound histone H4, nor were methylated arginine residues detectable upon incubating intact nuclei or chromatin with S-adenosylmethionine.     

Histone phosphokinase activity in nuclear and cytoplasmic cell fractions from normal and regenerating rat livers

1. Liver cell fractions were prepared by non-aqueous procedures and nuclei were also obtained in a hyperosmotic sucrose medium. Histone phosphokinase activity, assayed with histone F1 as substrate, was present in the soluble fraction of the cytoplasm and also bound on to the chromatin fraction of the nucleus. 2. The activity of the enzyme increased sixfold in nuclei from regenerating livers 22h after partial hepatectomy. 3. The enzyme bound in the nucleus was only marginally activated by 1mum-3':5'-cyclic AMP which stimulated the cytoplasmic soluble enzyme fourfold. 4. Nuclei prepared by the non-aqueous technique were also able to phosphorylate histones F2a and F3 and showed histone phosphatase activity with histone F1 phosphate as substrate.