Highly polymerized ribonucleic acid (RNA) was isolated from cytoplasmic extracts of rat and calf liver (1). In view of the morphological and biochemical heterogeneity of cytoplasm it was desired to establish the intracellular localization of the RNA. Cytoplasmic fractionation was carried out by differential centrifugation of sucrose homogenates of rat liver, and RNA was isolated from three fractions: mitochondria, microsomes, and supernatant. Nearly all the highly polymerized RNA of the cytoplasm was found in the microsome fraction.
EXPERIMENTAL
Livers from young male Wistar-strain rats of 150 to 200 gm. weight, fed a normal diet of Fox Food Blox and water ad libitum, were used.
The animals were decapitated and allowed to bleed freely. Immediately after excision, the livers were frozen in dry ice-acetone mixture 10 to 20 min. ; livers not used immediately were stored under dry ice refrigeration. Temperatures of 0 ยฐ to 8 ยฐ C. were maintained during the preparation and fractionation of the homogenates in order to minimize ribonuclease activity.
An International type SB, size 1 model centrifuge, equipped with an angle head containing lusteroid tubes of 100-ml. capacity, was used to sediment nuclei and mitochondria. A preparative ultracentrifuge equipped with a cooling system was used to sediment microsomes.
The livers in 100-gm. lots were passed while in a semi-frozen state through a meat grinder into 2.5 volumes of 0.25 molar sucrose, except for preparation 1 in which the sucrose used was 0.88 molar as advocated by Hogeboom, Schneider, and Pallade (2). The liver suspension was homogenized in a Waring Blend0r 3 times for 10 seconds each time, and the homogenate stirred at relatively low speed for 15 min. The suspension was centrifuged at 3,000 times gravity (calculated for the bottom of the tubes) for 50 min. to bring down a mixed sediment of whole cells, nuclei, and mitochondria; the sediment was suspended in 0.25 molar sucrose solution and centrifuged 3 times at 3,000 times gravity for 5 min. each time in order to remove whole cells and nuclei. The supernatant from the last centrifugation was centrifuged at 3,000