TAKESHI UTSUNOMIYA and JAY S. ROTH

STUDIES ON THE FUNCTION OF INTRACELLULAR RIBONUCLEASES V. Rbonuclease Actvty n Rbosomes and Polysomes Prepared from Rat Lver and Hepatomas TAKESHI UTSUNOMIYA and JAY S. ROTH From the Insttute of Cellular Bology, Unversty of Connectcut, Storrs. Dr. Utsunomya's present address s The Insttute for Cancer Research, Fox Chase, Pennsylvana ABSTRACT The RNase actvty and propertes of rbosome and polysomc preparatons from normal rat lver and some hepatomas havc been examned. Polysomc and rbosome preparatons from the Novkoff, McCoy MDAB, and Dunnng hcpatomas had consderably hgher specfc RNase actvty than correspondng preparatons from normal rat lvcr, Novkoff asctes, or Morrs 5123 hcpatomas. The optmum ph of the RNasc was approxmately 8.5 for all samples tested, and the samples showed no evdence of latent RNase actvty when treated wth 3 M sodum chlorde, EDTA, urea, or p-chloromcrcurbcnzencsulfonc acd. The RNase actvty appeared to be assocated prncpally wth breakdown products and/or subunts smaller than 80S. In the presence of Mg -~-~ ons, subunts could reaggrcgate to form monomer rbosomes ndstngushable from the natural products, but some of the reassocated rbosomes could contan RNase actvty whch had been bound to the smaller partcles. Smlar results were obtaned wth spermnc. In the hcpatomas, evdence was obtaned for thc prccxstcncc of consderable amounts of the smaller, RNase-contanng subunts n the ccll. When a small amount of crystallne bovne pancreatc RNase was added to partly dssocated rbosomes, the RNase was found only n assocaton wth the smaller subunts, and lttle or no enzyme was takcn up by rbosomes or polysomes. The results have led to the concluson that RNase s not a normal consttuent of the rbosome or polysome, but that RNase may becomc assocated wth these partculates f dssocaton and rcassocaton take place. Some mplcatons of these fndngs for the stablty of messenger RNA and for the mechansm of ts breakdown arc dscussed. INTRODUCTION It seems clear that any apprecable ntracellular hydrolyss of messenger RNA (mrna), soluble RNA (srna), or even rbosomal RNA (rrna) wll nterfere wth the orderly and effcent synthess of proten. It appears strange, therefore, that ntracellular rbonucleases (RNases) and other RNA-degradng enzymes are so wde- spread and are even found n assocaton wth the rbosomal structures whch are so ntmately nvolved n proten synthess. The probable answer to ths dlemma s that, under some condtons, t s necessary to dspose of presently operatng mrna (and possbly also srna and rrna) and replace t wth new and dfferent 395

messages or other RNA types. If ths s true, a functon of ntraceuular RNase becomes clear,.e., to degrade the unwanted RNA. The queston then becomes: Is there an orderly process by whch mrna can be destroyed at the requste tme and place, or s the process a random one dependng upon the concentraton and locaton of the RNase and possbly other factors as well? In ths paper, we have examned the lq, Nase actvty of rbosome and polysome preparatons from normal rat lver and several transplantable rat hepatomas of dfferent growth rates. We have also nvestgated varous factors affectng the RNase actvty of these preparatons n an effort to answer the basc queston posed above. We have reached the tentatve conclusons that RNase s not a normal consttuent of the rbosome or polysome and that there s no orderly controlled turnover of mrna or other RNA components of the cell. Intracellular RNase may, however, become assocated n a number of ways wth the rbosome, dependng on the random dssocaton and reassocaton of ths partculate, and to an extent condtoned by the quanttes of RNase and t(nase nhbtor n the cell, whch n turn may be controlled by a host of factors. Rbosomes that have RNase assocated wth them are abnormal and may degrade mrna wth whch they come n contact n a random manner; the amount of these rbosomes n the total rbosome populaton wll therefore determne the rate of breakdown of messenger RNA and possbly ts rate of synthess. MATERIALS AND METHOD~ The methods for obtanng the rbosome and polysome preparatons, the treatment of the tssues, and the buffers used have been descrbed n the prevous paper. PREPARATION OF ~ ~ METAL-FREE'' RNA USED AS SUBSTRATE FOR RNASE ASSAY : Schwarz RNA (42 g) was dssolved n 700 m of glass-dstlled water, and the ph was adjusted to 7.0. The soluton, was dalyzed aganst 5 changes of 2,5 volumes of 0,025 ~ EDTA (ph 7.0) for 24 hr and then aganst 5 changes of 5.0 volumes of water for 72 hr. The concentraton of RNA n the dalyzed soluton was determned by absorbance measurements at 260 m/z before and after the dalyss, and a 1% soluton was prepared and used for the assay below. RNASE ASSAY : In a fnal volume of 1.5 ml, the ncubaton mxture contaned 0.5 ml of enzyme sample, 0.5 nfl of buffer (0.05 M Trs ph 7.6 con- TABLE I Degradaton of Rbosomal RNA of Rat Hepatomas Durng RNase Assay Absorbancy Radoactvty at 260 m,~ Acd- Acd- Acdsoluble soluble nsoluble fracton fracton fracton McCoy MDAB rbosomes Before ncubaton 0.111 1770 186,000 After ncubaton 0.381 2010 188,000 Novkoff rbosomes Before ncubaton 0.109 2760 143,000 After ncubaton 0.296 3450 134,000 tanng 0.025 M KC1 and 0.005 M MgClz and 0.5 ml of 1% purfed RNA. The assay was run n duplcate when possble. The RNA was added last, and the ncubaton was carred out at 37 C for 60 mn. Then 1.5 ml of precptatng agent (1 ~ HC1 n 76/0/0 ethanol + 0.5% LaC13) was added, the mxture shaken well, and allowed to stand for 10 to 15 ran. It was then fltered through a 7 cm crcle of Whatman No. 42 flter paper, and a watch glass was used to cover the funnel. One ml of the clear fltrate was added to 9.0 ral of water n a test tube. The dluted fltrate was mxed well, and the absorbancy at 260 mt~ was determned. Approprate enzyme and tssue blanks were run. A unt of RNase actvty s defned as an absorbancy ncrement of 0.001, and the specfc actvty as unts per mllgram of proten. When desrable, a standard contanng 0.0015 ~g of crystallne pancreatc RNase (prepared n 0.1% gelatn soluton) was run under the same condtons. Unless Gtherwse stated, the assay was carred out at ph 7.6. PREPARATION OF P~-LABELED RIBOSOMES: One mc of carrer-free P~ was dssolved n 0.8 m of 0.9% salne, and 0.2 ml of ths soluton was njected ntrapertoneally nto several rats on the second and thrd day after the transplantaton of MDAB and Novkoff hepatomas. The anmals were sacrfced on the fourth day, and a rbosomal preparaton was obtaned from the tumors. In the RNase assay, the acd-soluble fracton was separated from the precptated materals by centrfugaton at 500g for 10 ran. The precptate was suspended n a known volume of water, and 0.9 ml of ths suspenson, and 0.2 ml of the acd-soluble fracton were plated on stanless steel planchets and dred. Counts were made n a wndowless gas-flow counter. The results are shown n Table I. 396 TI~IE,IOLmNAL OF CELL BIOLOGY VOLVME 29, 1966

~ 1.2 1.0 - % ~ ~ ~ 0.8 MDAB 04 Dunnng N0vk0ff ~= 0.6 0.4 /Novkoff asctes t-- / o t, I, [, I 7.0 8.0 9.0 10.0 ph FIOURE 1 Effect of ph on RNase actvty of varous polysome preparatons. Assay as descrbed n the text, except that the buffers dd not contan Mg ++ n ths experment. Trs-HC1 buffers, 0.05 ~t, were used. RESULTS AND DISCUSSION RNASE ASSAY AND ACTIVITY OF POLY- SOME PREPARATIONS: In early experments, some dffculty was encountered n achevng proportonalty between P-.Nase actvty and the amount of rbosome or polysome preparaton. Snce the absorbancy of acd-soluble materals obtaned on precptaton of the ncubaton mxture was lower n some cases than that of the blank, t appeared lkely that the hgh rbonucleoproten content of the samples was ntroducng coprecptaton or adsorpton errors. (For a dscusson of ths problem, see references 1-3; for some of the expermental results wth polysome and rbosome preparatons, see reference 3.) We found that reducng the amount of proten of the polysome preparaton to the followng lmts gave good proportonalty: Novkoff hepatoma, 2 to 20 /~g of proten per mlllter; Dunnng hepatoma, 20 to 100 /zg of proten per mlllter; McCoy MDAB hepatoma, 10 to 100 #g of proten per mlllter. Polysome preparatons obtaned from normal rat lver and Morrs 5123-D hepatoma usually gave neglgble RNase actvty, although a small but sgnfcant actvty was observed n some cases. Although consderable varablty was encountered from one hepatoma polysome preparaton to the next, normal lver and Morrs 5123-D hepatomas generally had lttle or no actvty, whereas the other hepatomas lsted n Table I always had hgh actvty. When tests were made for latent RNase actvty (4) by addton of p-chloromercurbenzenesulfonc acd (0.6 n~) to the polysomes over a range of ph values from 7.0 to 10.0, lttle evdence was obtaned for ths type of actvty. OPTIMUM ph: The optmum ph for RNase actvty assocated wth polysome preparatons from normal rat lver and varous hepatomas s llustrated n Fg. 1. Under the condtons used, the optmum ph was around 8.5, for all the samples. Fg. 1 also llustrates the relatve RNase actvty of the polysome preparatons from lver and from dfferent tumors, and t may be noted that the polysome preparaton from the Novkoff asctes hepatoma has RNase actvty n the same low range as the polysome preparatons from lver and Morrs 5123-D hepatoma. SOURCE OF ACID-SOLUBLE PRODUCTS : One mportant queston s the extent of the degradaton of rbosomal RNA durng ncubaton of a rbosomal preparaton wth yeast RNA n the RNase assay. Ths was tested by preparng rbosomes labeled wth p32 (see Methods). After ncubaton for 1 hr as descrbed n the secton on RNase assay, approxmately 99% of the radoactvty remaned wth the acd-nsoluble fracton; ths fact ndcates mmedately that the rbosomes were not sgnfcantly degraded to acd-soluble products by the RNase assocated wth them. Of course, t s possble that, n the absence of the yeast RNA, there would be a greater degradaton of rbosomal RNA, but evdence from other experments ndcates that even the tumor rbosome preparatons, whch have relatvely hgh RNase actvty, are remarkably stable. DISTRIBUTION IN A SUCROSE DENSITY GRADIENT AND RNASE ACTIVITY OF A POLYSOME PREPARATION OBTAINED FROM McCOY MDAB HE'PATOMA: Fg. 2 A rshows the dstrbuton of materal n a polysome preparaton from the McCoy MDAB hepatoma as well as the dstrbuton of RNase assocated wth ths preparaton. RNase actvty was detected only n fractons contanng degradaton products of rbosomes or partcles havng sedmentaton rates less than that of monomer rbosomes. It s not clear whether T. UTSUNOMIYA AND,~. S. ROTH Intracellular Rbonucleases. V 397

1.5 -- 80s A Control 1.( - I ---I-=: "'7-" ~1 0- N /\ o 1./(--) B Incubated wth EDTA ~>~ - '; 1.0 t 200 ~ 0.5 -- 1 100 5 0 15 20 25 30 ;ottom Fracton number Top FIOtmE ~ A Dstrbuton n a lnear sucrose densty gradent (5 to ~0% n TKM buffer) of RNase actvty and partculate materal n a polysome preparaton obtaned from McCoy MDAB hepatoma. Centrfuged for 2 hr at ~5,000 RPM n a Spnco SW 25 rotor. FmtmE 2 B A polysome preparaton obtaned from McCoy MDAB hepatoma frst treated wth 5 mm EDTA and ncubated for 20 mn at 37 C before centrfugaton. these small partcles were derved from rbosomes or were components n the supernatant fracton whch were brought nto the polysome fracton as contamnants. It seems unlkely that they are contamnants, snce they would not be expected to sedment through 0.5 and 2.0 M sucrose layers; and even f some dd so, they would be removed by the second centrfugaton that s employed. It appears reasonable to assume, therefore, that these smaller partcles are degradaton products of the rbosomes. If ths s true, then a further breakdown of rbosomes mght gve ncreased RNase actvty. Ths was tested by treatng the polysome preparaton wth 5 mm EDTA for 20 mn at 37 C and then by densty gradent centrfugaton through a gradent contanng 1 rnm EDTA. Fg. 2 B shows the dstrbuton of partculate materal and RNase actvty under these condtons. The treatment wth EDTA resulted n complete degradaton of the polysomes and rbosomes, but dd not uncover any more RNase actvty than was found n the control. Ths mples that there s no "latent" RNase n the rbosomes or polysomes of MDAB hepatoma, and that the RNase actvty assocated wth the polysome preparaton s not assocated drectly wth the polysomes but rather wth some subunt or degradaton product of the rbosomes. RNASE ACTIVITY ASSOCIATED WITH A RIBOSOME PREPARATION OBTAINED FROM MCCOY MDAB HEPATOMA: Snce the RNase actvty appeared to be assocated prncpally wth small partculates, a rbosome preparaton from McCoy MDAB hepatoma was allowed to stand for 1 wk n the cold to favor breakdown. To obtan maxmum possble separaton n the densty gradent, a thn layer of ths preparaton (0.95 ml contanng 0.25 mg proten) was lad over the gradent. In ths case, TKM buffer ph 8.5 was used, and the centrfugaton was carred out for 8, nstead of 4, hr. The results of ths experment are shown n Fg. 3. A large peak of monomer rbosomes remaned, 398 THE JOURNAL OF CELL BIOLOGY VOLUME 29, 1966

::L E 0.3 -N 0.4 -- ( Sedmentaton 80s -- 400 300 1 J~ N 0.2 _ I! _ / 5 I0 15 20 25 Fracton number 200 u FIGURE 3 Dstrbuton n a lnear sucrose densty gradent (5 to ~0% n TKM buffer 100 ph 8.5) of RNase actvty and pat~teulate materal n a rbosome preparaton obtaned from McCoy MDAB hepatoma. Preparaton had been allowed to stand n the cold for 0 1 wk before eentrfugaton. and varous degradaton products, probably consstng of subunts and smaller peces of rbonucleoproten, were found n a second peak ot low sedmentaton rate. Almost all the RNase was found assocated wth ths second peak. In addton to storage n cold, Mg4~-free, dstlled water as a method for breakdown of polysomes and rbosomes, EDTA may be used (see precedng paper for effects of EDTA on rbosomes and polysomes); smlar results have been obtaned usng EDTA wth both McCoy MDAB and Novkoff hepatoma rbosomes. In the same way, treatment of a rbosome preparaton wth 4 M urea or wth 3 M NaC1 (3) dd not release any addtonal RNase actvty n the case of McCoy MDAB or Novlkoff hepatomas, and n every nstance the enzyme actvty was assocated wth the breakdown products and subunts. TIIE EFFECT OF MG %-}" IONS ON RNASE ACTIVITY OF A RIBOSOMAL PREPARATION : A postmtochondral fracton was prepared from Novokoff hepatoma by homogenzng the tssue wth 2.5 volumes of 0.25 M sucrose soluton n TK buffer ph 7.6 and by centrfugng the homogenate at 15,000g for 15 ran n a Spnco Model L preparatve ultracentrfuge. The supernatant fracton was dvded nto 2 equal parts, and a soluton of magnesum acetate was added to one to gve a fnal concentraton of 5 mm. The 2 samples were layered over a sucrose densty gradent 0.7 to 2.0 M and centrfuged at 25,000 RFM for 20 hr. (The gradent for the Mgq-~-con - tanng sample also contaned 5 mm Mgq+.) The results are shown n Fg. 4. In the absence of added Mg -~- ons, the polysomes dssocated to form rbosomes and rbosomal subunts whch was shown n earler experments. In the control (Fg. 4 A), therefore, no boundary for polysomes was observed. Regon a contans soluble RNA plus smaller RNA fragments; regon b, rbosomal subunts havng sedmentaton coeffcents from 30 to 60S; regon c, monomer rbosomes of 70 to 80S; and fnally, regon d, polysomes. Practcally no RNase actvty was assocated wth monomer rbosomes n regon c n the control, and some sgnfcant actvty was detected whch was assocated wth the subunts of the rbosomes n regon b. In the presence of Mg -~-~ ons, the subunts were aggregated to form monomer rbosomes, and some of the rbosomes were clustered wth, presumably, stll exstng messenger RNA to form polysomes n regon d. Ths mples that all of the rbosomal preparatons used n the precedng experments contaned not only "natural" monomer rbosomes but also unnatural or artfactual rbosomes whch were aggregates of dssocated rbosomes, the aggregates beng produced by the added Mg -~ ons. When the rbosomal subunts were aggregated, the RNase actvty whch was assocated wth them was lost, presumably because t s now stercally hndered by the large bulk of the rbosome. EFFECT OF SPERMINE ON RNASE ACTIVITY IN RIBOSOMES: When an experment smlar to the above was carred out wth addton of 0.5 mm spermne, smlar and even more dramatc results were obtaned. Dssocated partcles were aggregated to a consderable extent, and the RNase actvty completely dsappeared (3). At the concentraton used, t was demonstrated that T. UTSV~OMIYA AND,]'. S. ROTIt Intracellular Rbonucleases. V 399

- d 2.0 (, Sedmentaton 500 A Control 1.5 c b a~- 1000 1.0 500 0.5 o 2.0 1.5 B Treate0w, th M< 2L 0"T, 1.0 0.5 Icl It a I - */*1 //1 - / I l I --I 500 0 5 10 15 20 25 30 Fracton number FIGURE 4 The effect of added Mg ++ on the dstrbuton, n a sucrose densty gradent, of postmtoehondral materms and RNase actvty prepared from Novkoff hepatoma. The condtons as well as the sgnfcance of the dfferent marked peaks are descrbed n the text. spermne dd not nhbt the actvty of bovne or gunea pg pancreatc RNase (5), nor dd t nhbt rat lver rbosomal RNase. The acton of spermne or added Mg ++ to aggregate the partcles suggests that f t s desred to preserve the ntegrty of polysomes by addng Mg ++ ons to the homogenzng medum, some unnatural 80S partcles (contanng RNase) are obtaned n the preparaton. If, on the other hand, t s desred to avod ths aggregaton by not addng Mg ++ ons, then t s not possble to obtan a polysome preparaton. In other words, under the usual condtons of solaton of these partculates, t s not possble to solate polysomes wthout contamnants of artfactual aggregated rbosomal subunts. It appears, n addton, that there must be a consderable amount of rbosomal subunts n ntact cells of Novkoff (and McCoy MDAB) hepatomas, snce t has been shown that these 80S rbosomes mantan ther ntegrty n the absence of added Mg ++ ons for 1 wk (n the cold). The rbosomal subunts n peak b n Fg. 4 A must, therefore, have preexsted n the cell, snce they would not be expected to be formed n 1 day. ADSORPTION OF PANCREATIC RNASE BY RIBOSOMES: Fg. 4 shows that hgh RNase actvtes were detected n the a regon. It s possble that the 80S rbosomes n ntact cells of Novkoff hepatoma do not contan RNase, but that the RNase present n the soluble fracton s adsorbed by the 80S rbosomes when they dssocate owng to changes n onc strength, Mg ++, or ATP concentraton n the cytoplasm. To obtan a further understandng of ths process, the nteractons between solated RNase and rbosomes were studed. 400 ThE JOURNAL OF CELL BIOLOGY VOLUME ~9, 1966

' Sedmentaton A 80s Control Treated wth pancreatc RNase T 1.0 -- 80s -- 1000 B t'~, t, I o o.5 -- 500 _~ / / / " I... [ I ". t 0 20 30 Fracton number ~f'p* I I I 0 lo 20 30 FIeuR~. 5 The effect of crystallne bovne pancreatc RNase on rbosomes prepared from Novkoff bepatoma. Rbosomes havng approxmately 0.9 mg proten n 0.45 ml of TK buffer ph 7.6 were treated wth 0.45 ml of an aqueous soluton of bovne pancreatc RNase contanng 1 ug of enzyme per mlllter. The mxture was kept n the cold for S0 ran before t was layered over a sucrose densty gradent (5 to ~0% made n the same TK buffer) and centrfuged at ~5,000 x~p~ for 8 hr n the SW 25 rotor. The control sample contaned 0.~5 ml of water n place of the RNase soluton. The rbosomes used n the experment were frst solated as a polysome preparaton. The polysome preparaton was then suspended n TK buffer whch dd not contan Mg ++ ons, and therefore the polysomes dssocated to form monomers and subunts. Ths technque of preparaton of rbosomes, nstead of the conventonal one descrbed n the Methods secton, was used, to mnmze the amount of rbosomal RNase ntroduced nto the rbosomal sample. Fg. 5 A shows that ths was successful. The amount of RNase assocated wth the subunts s much less than the amount ordnarly found. As descrbed below, ncubaton wth pancreatc RNase degraded the subunts to form smaller partcles; ths fact s ndcated by the shft of the absorbancy peak from fracton number 18 n Fg. 5 A to fracton number 23 n Fg. 5 B. The decrease n absorbancy of the 80S peak after the enzymc treatment s due to the movement of the dssocated subunts away from the 80S peak. All added pancreatc RNase appeared to be assocated wth the degraded rbosomal fragments, and no RNase actvty was found n fractons contanng 80S partcles. Recently, Hess and Horn (6) observed that bovne pancreatc RNase almost completely dssocated 50S subunts of Streptococcus pyrogenes and dd not at all affect the 80S partcles. The results reported n ths paper and those of Hess and Horn ndcate agan that the dssocaton of ntact rbosomes s a prerequste for sgnfcant enzymc attack on rbosomal RNA; ths ndcaton had been speculated earler by Shgeura and Chargaff (7). Several other nvestgators have noted the nsenstvty of bacteral, plant, or anmal cell rbonucleoproten to moderate amounts of RNase (8-10)~ Sekevtz (11) has demonstrated that calf lver rbosomes can absorb all crystallne RNase added to a suspenson of these partcles f the proper concentraton of enzyme s used. In the presence of very large amounts of RNase, enzyme may be adsorbed on ntact 70 or 80S rbosomes and may cause almost T. UTSVNOmYA AND J. S. ROTH IntraceUular Rbonucleaees. V 401

2.0f A ~ Sedmentaton E 1.5 N ~ 1.0 - L- N 0.5 //// f - // -- x,, /: 1500 too0 > 50O C : 10 20 30 Fractonumber r ] 1 I I 10 20 30 FIGURE 6 A Dstrbuton of rbosomal subunts and RNase actvty obtaned from the Novkoff hepatoma n the absence of added Mg ++ ons. Centrfugaton at ~5,000 ~PM for 10 h. n a Spneo SW ~5 rotor. FIouRE 6 B Dstrbuton of rbosomal subunts and RNase actvty obtaned from Novkoff hepatoma n the absence of added Mg ++ ons after treatment wth hgh-speed supernatant fracton from the same source. Condtons the same as descrbed for Fg. 6 A. complete breakdown of the partcles (12). Neu and Heppel (13) also demonstrated that, under certan condtons, larger partcles of E. col (70S, 100S) could absorb RNase. ABSORPTION OF SUPERNATANT FRACTION RNASE BY A PREPARATION OF RIBOSOMES OBTAINED FROM I-IEPATOMA : The absorpton of bovne pancreatc RNase by rbosomal subunts suggests that RNase present n the supernatant fracton of Novkoff hepatoma mght also be adsorbed by rbosomes or rbosomal subunts solated from the same source. To test ths possblty, a rbosomal preparaton was obtaned n TK1V[ buffer, and the fnal pellet was suspended n TK buffer, ph 7.6, by homogenzng the partcles n a Ten Broeck homogenzer. To nsure some breakage of the 80S partcles to smaller unts, we appled about 15 strokes. Ths was favored also by lack of added Mg "~+. Ths rbosome fracton and the supernatant fracton from Novkoff hepatoma were ncubated together at 4 C for 10 ran and then centrfuged for 10 hr at 25,000 RPI~. The prolonged centrfugaton tme was employed to avod overlap of peaks f possble. In the control run, Fg. 6A, all rbosomal partcles were sedmented at the lower part of the centrfuge tube, and only a modest amount of l~nase actvty was assocated wth them. These partcles were degraded rapdly durng 10 ran of ncubaton and durng subsequent contact wth the supernatant fracton as shown n Fg. 6 B. The amount of materals absorbng at 260 m/~ at the lower part of the tube decreased remarkably, and RNase actvty was detected throughout the tube. The enzyme appears to be bound to the dssocatng rbosomes; f t were not bound and exsted n free form, t could not sedment below fracton 18 to 20 under the condtons used n ths centrfugaton. ADSORPTION OF SUPERNATANT FRACTION RNASE BY A POLYSOME PREPARATION : When smlar experments were carred out wth a polysome preparaton from Novkoff hepatoma, t was found that the polysomes and rbosomes were essentally unaffected by ths treatment. Some of the RNase actvty of the supernatant fracton dd bnd, however, to monomer and dmer unts, but the largest porton was bound to subunts as observed wth other systems. GENERAL DISCUSSION AND CONCLUSIONS Intraccllular RNase n the lver cell probably does not exst free, but s, for the most part, bound by RNase nhbtor and, to a small extent, attachcd to rbosomal subunts. Under normal condtons, therefore, t would not bc expected to attack mrna, ntact 80S rbosomes, or polysomes, and RNase does not appear to bc a normal structural component of the latter two partculates. 402 TH~ JOURNAL OF CELL BIOLOGr VOLUME 29, 1966

Under some condtons, dssocaton and reassocaton of rbosomal partcles and subunts may take place (14). Dssocaton would be favored by absence of Mg salts and agents whch, generally, complex Mg ~ on. Reassocaton would be favored by the reverse of these condtons or by addton of spermne. Occasonally, a rbosome wll be reformed from a subunt whch contans bound RNase. The 80S partcle so formed would be ndstngushable from the "natve" 80S rbosome but would contan RNase. The RNase would be prevented, probably by stereochemcal hndrance, from attackng the rbosome, although t may attack yeast RNA. The artfactual rbosome may become attached to an mrna strand and, n ths case, snce the locaton of the RNase s probably close to that of the attachment of the mrna strand, the strand may be cut by the enzyme. Thus, the breakdown of mrna n the lver cell s seen as a process occurrng by chance; the extent and rapdty of the breakdown depends on the concentraton of artfactual rbosomes. Ths, n turn, obvously depends upon the concentraton of RNase as well as that of RNase nhbtor. In the lver, the RNase concentraton s low and nhbtor concentraton s hgh, and therefore t would be expected that the concentraton of artfactual rbosomes would be low and the breakdown of mrna a rare event. Ths s supported by some expermental observatons whch suggest that mrna n lver s relatvely stable (15), although there are a number of reports to the contrary. Rapdly prolferatng hepatomas such as Dunnng, Novkoff, and McCoy MDAB have much hgher RNase actvty assocated wth ther rbosomes and polysomes than normal rat lver or the slowly growng Morrs 5123-D hepatoma. The rapdly prolferatng hepatomas also contan amounts of RNase nhbtor much lower than normal (16). Thus, they undoubtedly contan a hgher proporton of artfactual rbosomes wth subsequently more rapd destructon and turnover of ther mrna, and ths may account, n part, for the preponderance of monomer and dmer unts observed n these tumors. (See prevous paper and reference 17.) Fnally, the recent work of Neu and Heppel (13, 18, 19) on E. col rbosomal RNase has led to somewhat smlar conclusons wth respect to ths dfferent system. Ths paper was supported by grants from the Natonal Cancer Insttute, Natonal Insttutes of Health, CA-07824, and the Amercan Cancer Socety, P-303. Dr. Roth was supported by Research Career Award, RC 31-63, from the Natonal Cancer Insttute, Natonal Insttutes of Health. Ths paper was abstracted from Dr. Utsunomya's thess presented n partal fulfllment of the requrements for the Ph.D. degree, Unversty of Connectcut, 1965. Receved for publcaton 28 October 1965. BIBLIOGRAPHY 1. LEPOUTRE, L., STOCKX, J., and VANDENDRIES- SCHE, L., Anal. Bochem., 1963, 5, 149. 2. FIERS, W., and M~ILLER, K. M., Compt. rend. tray. Lab. Carlsberg, 1960, 31, 507. 3. ROT, J. S., n Advances n Cancer Research Methods, (H. Busch, edtor), New York, Academc Press Inc., n press. 4. ROTH, J. S., J. Bol. Chem., 1958, 231, 1097. 5. SIEKEVITZ, P., and PALADE, C. E., J, Cell Bol., 1962, 13, 217. 6. HESS, E. L., and HORN, R., J. Mol. Bol., 1964, 10, 541. 7. SHIGEURA, H. T., and CHARGAFF, E., Boehm. et Bophyszca. Acta, 1960, 37, 347. 8. TlSSI~RES, A., and WATSON, J. D., Nature, 1958, 182, 778. 9. Ts'o, P. O. P., BONNER~ J., and VINOOR.~d), J., Bochm. et Bophysca Acta, 1958, 30, 570. 10. ARNST~IN, H. R. V., Bochem. J., 1961, 81, 24P. 11. SIEK~VITZ, P., Ann. New York Acad. Sc., 1963, 103, 773. 12. ROTH, J. S., J. Bophysc. and Bochem. Cytol., 1960, 7, 443. 13. NEu, H. C., and HEPPEL, L. A., Proc. Nat. Acad. Sc., 1964, 51, 1267. 14. HOSOKAWA, K., FUjIMURA, R. K., and NOMORA, M., Proc. Nat. Acad. Sc., 1966, 55, 198. 15. REVEL, M., and HIATT, H. H., Proc. Nat. Acad. Sc., 1964, 51, 810. 16. ROTH, J. S., HILTON, S., and MORRIS, H. P., Cancer Research, 1964, 24, 294. 17. WEBB, T. E., BLOBEL, G., and POTTER, V. R., Cancer Research, 1964, 24, 1229. 18. NEU, H. C., and HEPPEL, L. A., Bochem. and Bophysc. Research Commun., 1964, 14, 109. 19. NEU, H. C., and HEPPEL, L. A., Bochem. and Bophysc. Research Commun., 1964, 17, 215. W. UTSUNOM/YA AND J. S. ROTIt Intracellular Rbonucleases. V 403