Studies on Ribonucleic Acid from Rat Liver Ribosomes

In most, ribosomes, of whatever origin, the total amount, of ribonucleic acid is close to 1,800,OOO molecular weight units (1).How many separate pieces of RNA, either structural or informational, are present, and how large these pieces are, however, has not been definitely established.The great variation in the reported sedimentation coefficients of ribosomal RNAs (1) may be ascribed to two principal causes: the presence of ribonucleases and other enzymes which lead to breakage of the phosphodiester bonds, and the tendency of RNA fragments to be linked together by traces of protein, by dibasic metals, and by hydrogen bonds.This paper describes the isolation of the ribosomal RNA of rat liver cytoplasm by a method that attempts to avoid the first, of these difficulties by the use of bentonite and other ribonuclease inhibitors, and by emphasis on speed and low temperatures.The second difficulty has been dealt with by examining the RNA under conditions where aggregation should be minimal.After conversion to the sodium salt,, and the breaking of interchain hydrogen bonds, the major component of the RNA has a molecular weight of about, 550,000. EXPERIMENTAL PROCEDUREBe&mite-Bentonite was equilibrated with buffer containing 0.001 M potassium phosphate and 0.0005 M magnesium chloride, pH 7, and fractionated as described previously (2).The fraction sedimenting between 8,000 X g and 20,000 X g in 15 minutes is designated coarse magnesium bentonite; the fraction sedimenting between 20,000 X g and 78,000 X g, in 30 minutes, is called fine magnesium bentonite.Coarse sodium bentonite was prepared by dialyzing the coarse magnesium bentonite against 0.01 M sodium phosphate-O.001M EDTA, pH 7.Ribosomes-Adult male rats were fasted overnight.In groups of three, they were heavily anesthetized with ether, and their livers were removed and buried in crushed ice.All subsequent steps were carried out at, 2-5", except where noted.The tissue was forced through a mincer (2) and 30 g of pulp were suspended in 150 ml of 0.3 M sucrose in a pestle homogenizer.The suspension was then homogenized in the micro attachment of a Waring Blendor for 3 minutes at 40 volts.Fine magnesium bentonite, 15 mg, and 1500 units of penicillin were added to the blendor.The cell debris, nuclei, and mitochondria were removed by centrifugation for 5 minutes at 20,000 X g in the No. 30 rotor of the Spinco model L ultracentrifuge.The superna-