Free Amino Acids and Nucleic Acid Content of Cell Nuclei Isolated by a Modification of Behrens' Technique

Abstract

It has already been found that a considerable number of enzymes occur in nuclei of rat liver cells which have been isolated at pH 6.0 by a procedure involving the use of the Waring blendor with very dilute citric acid for breaking the cells, followed by differential centrifugation (1-3). As a complementary type of work, studies have been started to determine the relative concentrations of some important substrates in the cell nucleus as compared to the corresponding concentrations in the whole cell or in the cytoplasm. Since many of the substrates of the cell are water-soluble it was necessary to avoid aqueous solutions in the preparation of the nuclei. For this purpose a modification of the method of Behrens (4), which employs only non-polar organic solvents, was developed. The free amino acid pattern of these nuclei was determined by paper partition chromatography, and their nucleic acid content estimated. The method of Behrens has already been used in a study of the distribution of vitamins between nucleus and cytoplasm (5). EXPERIMENTALA. Source of Material.--Wistar strain rats fed ad libitum on a "fox chow" diet were killed by decapitation. The livers were removed as quickly as possible, easily visible fibrous tissue was removed, and the livers were then immediately frozen by being placed in beakers cooled in an acetone-dry ice bath. The frozen livers were allowed to stand at -15 ° in a cold room for about an hour, and then were broken into as small pieces as possible. The resulting material was then immediately lyophilized continuously during an interval of 36 to 48 hours. When nearly all the water had been removed, the material was powdered with a mortar and pestle and lyophilization was continued for 5 to 6 hours. The final powder was immediately used in the cell fractionation procedure described below. B. Preparation of Cell Nuclei, Whole Cells, and the Cytoplasmic Fractions.--Fivepreparations of nuclei were isolated from liver powder lyophilized as above, three from rat liver and two from rabbit liver. Fifty gin. of the lyophilized powder were ground in a ball mill of 1 liter capacity at -15 ° for 24 hours in the presence of 200 ml. of 50-4)0 ° petroleum ether with about 160 irregular pebbles of 15 to 20 ram. average diameter. The petroleum ether facilitated grinding so that relatively small proportions of unbroken cells remained even though the grinding time was not prolonged. The 629on May 12, 2018 jgp.rupress.org Downloaded from

The deoxyribonucleic acid content of the rat cell nucleus and its use in expressing the results of tissue analysis, with particular reference to the composition of liver tissue

“…I1 liver tissue after removal of tumour nodules. DISCUSSION Our values for the average DNAP content/nucleus in normal adult rat liver (Table 1) are in fairly good agreement with those obtained by other workers using similar methods (Dounce, Tishkoff, Barnett & Freer, 1950;Ely & Ross, 1951a;Harrison, 1951;Leuchtenberger et al 1951;Campbell & Kosterlitz, 1952;and Leuchtenberger et al 1952). To calculate the DNA content/liver nucleus Price and his coworkers determined the amount of DNA/100 g. fresh liver and divided this figure by the number of nuclei/100 g., estimated by nuclear counts on a homogenate of a weighed portion of tissue (Price, Miller, Miller & Weber, 1950;Price & Laird, 1950).…”

Electron Microscopic and Biochemical Characteristics of Nuclei and Nucleoli Isolated From Rat Liver

“…Nuclei then sediment towards the bottom of the tube during centrifugation, but lighter components, such as cytoplasmic organelles, whole cells, erythrocytes, and nuclei with tabs of cytoplasm float to the surface. This principle was first applied in nonaqueous procedures (8)(9)(10)(11) and was subsequently adapted to aqueous methods by the use of 2.2 M sucrose (12)(13)(14)(15)(16)(17)(18)(19)(20). These methods appeared to give satisfactory preparations of nuclei, although the yields were often low (16,17) and the methods were difficult to adapt to the processing of large quantities of tissue.…”

Intracellular Localization of Type 4 Adenovirus

“…The technique involving mechanical disruption of cells and repeated washings with sucrose solutions was adopted after preliminary studies with adenovirushifected HeLa cells showed that this procedure gave better results than other methods tested. Several other techniques were discarded when it was found that the yield of nuclei was too low or that adenovirus was inactivated during the procedure (18)(19)(20)(21). The method employed was originally described by Schneider and Hogeboom (13) and used successfully by Ackermann and Kurtz (22) and Gray and Scott (14) in studies of the intracellular localization of herpes simplex virus in the liver of the mouse or chick embryo.…”