V. Ribonuclease Activity in Ribosomes and Polysomes Prepared from Rat Liver and Hepatomas

The RNase activity and properties of ribosome and polysomc preparations from normal rat liver and some hepatomas havc been examined. Polysomc and ribosome preparations from the Novikoff, McCoy MDAB, and Dunning hcpatomas had considerably higher specific RNase activity than corresponding preparations from normal rat livcr, Novikoff ascites, or Morris 5123 hcpatomas. The optimum pH of the RNasc was approximately 8.5 for all samples tested, and the samples showed no evidence of latent RNase activity when treated with 3 M sodium chloride, EDTA, urea, or p-chloromcrcuribcnzencsulfonic acid. The RNase activity appeared to be associated principally with breakdown products and/or subunits smaller than 80S. In the presence of Mg -~-~ ions, subunits could reaggrcgate to form monomer ribosomes indistinguishable from the natural products, but some of the reassociated ribosomes could contain RNase activity which had been bound to the smaller particles. Similar results were obtained with sperminc. In the hcpatomas, evidence was obtained for thc prccxistcncc of considerable amounts of the smaller, RNase-containing subunits in the ccll. When a small amount of crystalline bovine pancreatic RNase was added to partly dissociated ribosomes, the RNase was found only in association with the smaller subunits, and little or no enzyme was takcn up by ribosomes or polysomes. The results have led to the conclusion that RNase is not a normal constituent of the ribosome or polysome, but that RNase may becomc associated with these particulates if dissociation and rcassociation take place. Some implications of these findings for the stability of messenger RNA and for the mechanism of its breakdown arc discussed. I N T R O D U C T I O N It seems clear that any appreciable intracellular hydrolysis of messenger RNA (mRNA), soluble RNA (sRNA), or even ribosomal RNA (rRNA) will interfere with the orderly and efficient synthesis of protein. It appears strange, therefore, that intracellular ribonucleases (RNases) and other RNA-degrading enzymes are so widespread and are even found in association with the ribosomal structures which are so intimately involved in protein synthesis. The probable answer to this dilemma is that, under some conditions, it is necessary to dispose of presently operating mRNA (and possibly also sRNA and rRNA) and replace it with new and different 395 on O cber 0, 2017 jcb.rress.org D ow nladed fom messages or other R N A types. If this is true, a function of intraceUular RNase becomes clear, i.e., to degrade the unwanted RNA. The question then becomes: Is there an orderly process by which m R N A can be destroyed at the requisite time and place, or is the process a r andom one depending upon the concentrat ion and location of the RNase and possibly other factors as well? In this paper, we have examined the lq, Nase activity of ribosome and polysome preparations from normal rat liver and several transplantable rat hepatomas of different growth rates. We have also investigated various factors affecting the RNase activity of these preparations in an effort to answer the basic question posed above. We have reached the tentative conclusions that RNase is not a normal constituent of the ribosome or polysome and that there is no orderly controlled turnover of m R N A or other R N A components of the cell. Intracellular RNase may, however, become associated in a number of ways with the ribosome, depending on the random dissociation and reassociation of this particulate, and to an extent conditioned by the quantities of RNase and t(Nase inhibitor in the cell, which in turn may be controlled by a host of factors. Ribosomes that have RNase associated with them are abnormal and may degrade m R N A with which they come in contact in a random manner ; the amount of these ribosomes in the total ribosome population will therefore determine the rate of breakdown of messenger R N A and possibly its rate of synthesis. M A T E R I A L S AND M E T H O D ~ The methods for obtaining the ribosome and polysome preparations, the treatment of the tissues, and the buffers used have been described in the previous paper. P R E P A R A T I O N O F ~ ~ M E T A L F R E E ' ' R N A USED AS SUBSTRATE FOR RNASE ASSAY : Schwarz RNA (42 g) was dissolved in 700 mi of glass-distilled water, and the pH was adjusted to 7.0. The solution, was dialyzed against 5 changes of 2,5 volumes of 0,025 ~ EDTA (pH 7.0) for 24 hr and then against 5 changes of 5.0 volumes of water for 72 hr. The concentration of RNA in the dialyzed solution was determined by absorbance measurements at 260 m/z before and after the dialysis, and a 1% solution was prepared and used for the assay below. RNASE ASSAY : In a final volume of 1.5 ml, the incubation mixture contained 0.5 ml of enzyme sample, 0.5 nfl of buffer (0.05 M Tris pH 7.6 conT A B L E I Degradation of Ribosomal RNA of Rat Hepatomas During RNase Assay Absorbancy Radioactivity at 260 m,~ AcidAcidAcidsoluble soluble insoluble fraction fraction fraction McCoy MDAB ribosomes Before incubation 0.111 1770 186,000 After incubation 0.381 2010 188,000 Novikoff ribosomes Before incubation 0.109 2760 143,000 After incubation 0.296 3450 134,000 taining 0.025 M KC1 and 0.005 M MgClz and 0.5 ml of 1% purified RNA. The assay was run in duplicate when possible. The RNA was added last, and the incubation was carried out at 37°C for 60 min. Then 1.5 ml of precipitating agent (1 ~ HC1 in 76/0/0 ethanol + 0.5% LaC13) was added, the mixture shaken well, and allowed to stand for 10 to 15 rain. It was then filtered through a 7 cm circle of Whatman No. 42 filter paper, and a watch glass was used to cover the funnel. One ml of the clear filtrate was added to 9.0 ral of water in a test tube. The diluted filtrate was mixed well, and the absorbancy at 260 mt~ was determined. Appropriate enzyme and tissue blanks were run. A unit of RNase activity is defined as an absorbancy increment of 0.001, and the specific activity as units per milligram of protein. When desirable, a standard containing 0.0015 ~g of crystalline pancreatic RNase (prepared in 0.1% gelatin solution) was run under the same conditions. Unless Gtherwise stated, the assay was carried out at pH 7.6. PREPARATION OF P~-LABELED R I B O S O M E S : One mc of carrier-free P~ was dissolved in 0.8 mi of 0.9% saline, and 0.2 ml of this solution was injected intraperitoneally into several rats on the second and third day after the transplantation of MDAB and Novikoff hepatomas. The animals were sacrificed on the fourth day, and a ribosomal preparation was obtained from the tumors. In the RNase assay, the acid-soluble fraction was separated from the precipitated materials by centrifugation at 500g for 10 rain. The precipitate was suspended in a known volume of water, and 0.9 ml of this suspension, and 0.2 ml of the acid-soluble fraction were plated on stainless steel planchets and dried. Counts were made in a windowless gas-flow counter. The results are shown in Table I. 396 TI~IE ,IOLmNAL OF CELL BIOLOGY • VOLVME 29, 1966 on O cber 0, 2017 jcb.rress.org D ow nladed fom