The metabolism of isolated rat-liver nucleoli and other subnuclear fractions. The active site of amino acid incorporation in the nucleus

Abstract

It was shown by Rees & Rowland (1961) that rat-liver nuclei isolated in 025 M-sucrose incorporated amino acids into protein, and nucleotides into RNA. Although the incorporation was inhibited to varying degrees by anoxia and by several inhibitors of oxidative phosphorylation, it was not affected by detergents, by freezing and thawing the nuclei, or by disruption of the nuclei by ultrasonic vibration (Rees, Ross & Rowland, 1961). We have now studied isolated nucleoli and other subnuclear fractions in order to investigate the active site of synthesis of proteins and nucleic acids within the nucleus. In addition the enzymic and chemical composition of the various fractions has been examined. Part of this work has been published in a preliminary form (Rowland, Rees & Varcoe, 1962). MATERIALS AND METHODS Animals. Male albinorats,weighing200-250g., were used. Reagent8. These were as described by Rees & Rowland (1961). In addition [1-34C]valine and [1-14C]leucine were obtained from The Radiochemical Centre, Amersham, Bucks. Nuclear and8ubnuclearpreparation&. Nuclei were isolated from rat liver in 0*25M-sucrose as described by Rees & Rowland (1961). Scheme 1 shows the method used for isolating nucleoli and other subnuclear fractions. Batches of isolated nuclei, suspended in 15-20 ml. of 0 25m-sucrose, were subjected to ultrasonic vibration (20 keyc./sec.) at 20 in a MSE ultrasonic disintegrator (60w) with a titanium probe of 1 cm. diam. until all nuclei were disrupted (usually 7 min.). The resulting suspension was centrifuged for 30 sec. at 2500g to remove probe debris and coagulated protein. The nucleoli I Sediment Recentrifuged twice in water (see text) Nucleoli (fraction A) Disrupted nuclei Centrifuged at 412 x 104g,^min.

Intranuclear Changes in Rat Liver During the Early Stages of Feeding the Hepatocarcinogens Thioacetamide and 4-Dimethylaminoazobenzene

“…Since the onset of histological changes is delayed in comparison with thioacetamide (Rees and Rowland, 1961) the time intervals for killing were 14, 21 and 35 days. Fig.…”

“…Chemical determinations.-Ribonucleic acid (RNA) and phospholipids were determined as described previously (Rees et al, 1963). Protein nitrogen was determined by the method of Lowry, Rosebrough, Farr and Randall (1951).…”

“…Although the main site of binding of these compounds is cytoplasmic, some nuclear binding also takes place (Magee, 1962). Studies on pre-cancerous liver have revealed marked nuclear changes both histologically and biochemically at early stages (Rees and Rowland, 1961) and the question arises whether these changes are the result of the small degree of interaction between nucleus and carcinogen or whether they are secondary to the interaction of carcinogen with cytoplasmic components. In these studies it was found that there was a marked increase in both nuclear RNA and phospholipid.…”

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IT has been previously shown that during the feeding to rats of thioacetamide and 4-dimethylaminoazobenzene (DAB), parenchymal liver cell nuclei show an increase in their chemical constituents (Rees and Rowland, 1961). These measurements were carried out at regular time intervals throughout the tumour induction period of approximately 6 months.

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“…As a comparison, similar studies have been made on the livers of rats fed with DAB for up to 5 weeks, no histological changes being apparent before 3 weeks with this carcinogen (Rees and Rowland, 1961). In addition to the determination of chemical composition of these fractions the incorporation in vivo has been followed of 32P into ribonucleic acid (RNA) and phosphotlipid.…”