Nuclear preparations

Castro GD, Diaz-Gomez Ml, Castro JA. 1990. Biotransformation of carbon tetrachloride and lipid peroxidation promotion by liver nuclear preparations from different animal species. Cancer Lett 53 9-15. 

D. Conversion of l-Glutamate to Pyrrolidone Carboxylate by Rat Liver Nuclear Preparations... 

Although glucose-6-phosphatase activity in the nuclear fraction which has generally been observed may in part result from contamination of nuclear preparations with unruptured cells and debris, activity has also been observed with purified nuclei (62). Further, Kashnig and Kasper (63) have recently identified glucose-6-phosphatase as a component of rat liver nuclear membrane. 

PolyPs with an average chain length of 100 residues were observed in the nuclei of Physarium polycephalum (Pilatus et al, 1989). Nuclear preparations of mammalian cells were found to be relatively enriched in PolyPs (Kumble and Kornberg, 1995 Kornberg, 1999). 

Why are elements of odd Z more satisfactory targets for nuclear preparations than those of even Z )... 

Gunasekera, N., Xiong, G, Musier-Eorsyth, K., and Arriaga, E., A capillary electrophoretic method for monitoring the presence of alpha-tuhuhn in nuclear preparations. Anal. Biochem., 330, 1—9, 2004. 

For isolation of nucleoli, 10 ml of the nuclear preparation is placed in a 20-ml sonication rosette flask (Heat Systems) and sonicated on ice for three to four bursts of 30 sec each to disrupt the nuclei and release nucleoli. The efficiency of sonication is monitored by Azure C staining. 

Further experiments have shown that an RNA fraction obtained from Xenopus gastrulae by treatment of a crude nuclear preparation (which... 

Hayaishi and co-workers (149) were the first to observe that when a nuclear preparation from rat liver was incubated with radiolabeled NAD, the resulting newly synthesized poly(ADP-ribose) was associated with histones Hi, H2A, H2B, and H3 (149). As summarized in the previous section, other groups have corroborated this observation and extended it to include nonhistone nucleosomal proteins (2, 64, 84, 90, 164,178,179, 200, 215, 229). Differences in techniques for isolation of poly ADP-ribosylated proteins and the presence of poly(ADP-ribose) degradative enzymes [poly(ADP-ribose) glycohydrolase and various phosphodiesterases] may explain some of the conflicting reports as to primary acceptor protein, extent of modification, and length of polymer. 

Activities were measured in a crude nuclear preparation and are expressed as pmol/min/10 cells (mean SD)... 

Hewish DR, Burgoyne LA (1973) The calcium dependent endonuclease activity of isolated nuclear preparations. Relationship between its occurence and the occurence of other classes of enzymes found in nuclear preparations. Biochem Biophys Res Commun 52 475-781... 

The stimulation of ADP-ribosylation of proteins associated with both pertussis toxin and cholera toxin involves modification of plasma membrane proteins [5—7]. To localize the site of maximal ADP-ribosylation of cellular proteins after exposure to cAMP, isolated nuclear preparations were simultaneously compared to whole cell preparations. Nuclei were isolated by a nonionic detergent method [14]. 

Fig. 1 Immunoblots of acid extracts of nuclear preparations from mouse epidermal cells JB6 (clone 41) which had been treated with active oxygen produced by xanthine/xanthine oxidase (50 pg/ml xanthine plus 5 Xg/ml xanthine oxidase) for 30 min or 5 pg/ml MNNG for 20 min. The extracts were purified on a boronate affinity column and the proteins separated on 15% polyacrylamide gels (34). A polyclonal rabbit antibody against histone H3 (37) was used for the immunoblot and the blot was reacted with [i Sjj-iabeled donkey anti-rabbit IgG. Densitometer scannings are shown at the right side of the autoradiogram and were obtained with a Zeineh "soft laser" scanning densitometer.

Fig. 1-2. Cellular organelles obtained by tissue fractionation. Electron micrograph of the nuclei (upper left), mitochondria (lower right), microsomes (upper right), and zymogen granules (lower right) prepared by differential centrifugation. The nuclear preparation picture was provided by Palade, the others were prepared in collaboration by Van Lancker and Swift...

Busch and his associates [18-21] attempted to isolate nucleoli from either Walker tumors or liver treated with thioacetamide. They identified the nucleoli with the aid of azure and claim to have obtained, after sonication of the nuclei followed by differential centrifugation, a nucleolar preparation 90-95% free of contamination. The preparation is rich in DNA, relatively poor in RNA but because the yield in nucleoli is low, their nucleolar preparation can hardly be considered representative of the original nuclear preparation. 

Among all particulate fractions of the cells, the nucleus is most likely to suffer leakage during tissue fractionation. As pointed out before, in contrast to mitochondria, the nucleus is not surrounded by a continuous membrane, but rather by a perforated envelope. As with any other particulate preparation, it is difficult to evaluate the amount of material that has been adsorbed onto the nucleus. Deoxyribonucleic acid and ribonucleic acid are both apt to adsorb proteins therefore, it would not be surprising if proteins that are sometimes considered genuine constituents of the nucleus remain associated with the nuclear preparation only because they are artifacts of the preparative procedure. 

Biochemists have demonstrated at least 80 different enzymes in preparations of nuclei. Among them are enzymes of the main bioenergetic pathways and a large number of hydrolytic enzymes. On the basis of their relationship to the nucleus, the enzymes in nuclear preparations can be divided into three different groups (1) those likely to be contaminants (2) those clearly related to the nucleus and (3) those present in the nucleus. 

The succinoxidase and cytochrome oxidase found in the nuclear pellet obtained by tissue fractionation are probably not normal constituents of the nucleus. The activities associated with nuclei obtained by tissue fractionation are proportional to the number of mitochondria contaminating the preparation. No appreciable amount of cytochrome c or flavoprotein is found in the nuclear preparation. Therefore, it seems that... 

The presence of glycolytic enzymes in the nuclear preparation does not prove that those enzymes are normal constituents of the nucleus in the intact cell. Much more sophisticated and up-to-date investigations are required to demonstrate such an association beyond a reasonable doubt. To our knowledge, such investigations have been attempted only in the case of aldolase. 

Roodyn [34] studied the distribution of aldolase in liver nuclear preparations centrifuged in a gradient concentration of sucrose. He demonstrated that although the distributions of aldolase and DNA were similar, cytochrome oxidase and succinoxidase were found in particles of different sizes. His results suggested that aldolase and DNA are closely associated. The possibility of contamination was eliminated in experiments in which aldolase was added to the nuclear preparation because the added aldolase, in contrast to the aldolase originally associated with the nuclear pellet, could be easily washed off. 

Nuclear oxidative phosphorylation is difficult to quantify. Although oxygen uptake in the nucleus can be measured, no exact P/O ratio is available. This is because only the AMP already present in the nuclear preparation can be converted to ATP any AMP added to the nuclei remains unaltered. An intriguing observation is the effect of DNase on the phosphorylation of AMP. (Allfrey has proposed that DNase blocks ATP synthesis in the nucleus indirectly namely, by inhibition of the nuclei by the histones, which after DNA extraction are no longer associated with DNA by salt linkages [35].) The enzymic extraction of 55% of the DNA in the nucleus leads to the loss of nuclear phosphorylation properties, which can be restored by adding DNA to the system. The effect of DNA is not specific because DNA can be replaced by RNA, polyadenylic acid, heparin, chondroitin sulfate, and polyethylene sulfate. Oxidative phosphorylation in thymus nuclear preparation has been confirmed in two laboratories. Whole body doses of ionizing radiation inhibited oxidative phosphorylation in thymus nuclei. 

Klouwen and his associates [38, 39] have further investigated the occurrence of oxidative phosphorylation in thymus nuclei they acknowledge contamination of the nuclear pellet with as much as 10% intact cell. Yet they believe that the small levels of oxidative phosphorylation measured in nuclei (P/O ratio 0.6-1.0) do not result from contamination by intact or fragmented mitochondria. Further, they claim that the nuclear oxidative phosphorylation is sensitive to various inhibitors of the electron transport chain. Using low-temperature spectophotometry, it was established that a nuclear preparation free of cytoplasmic contamination contained cytochrome b, c, and aa3 and that as much as 40% of cytochrome aa3 may be found in association with calf thymus nuclei. Other researchers have also found cytochrome b and c, but not cytochrome aa3, in calf thymus nuclear preparations. 

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