RNase Activity in Erythroid Cell Lysates

EDWARDR. BuPKA

From The Cardeza Foundation for Hematologic Research, Department ofMedicine, Jefferson Medical College, Philadelphia, Pennsylvania 19107

A B S T R A C T The characteristics of degradation ofreticulocyte ribonucleic acid (RNA) and ribosomeswere studied in a whole erythroid cell lysate system.The process followed Michaelis-Menten kinetics, andindicated that RNA degradation in the erythroid cellis mediated by an enzyme previously isolated from reticu-locyte hemolysates. Erythroid cell RNase activity had atemperature optimum of 500C, a pH optimum of 7.0, wasnot energy dependent, was heat labile at physiologic pH,and was inhibited by Mg", Ca", and exposure to ben-tonite and deoxycholate. Free sulfhydryl groups werenot essential for RNase activity. Of the substrates oc-curring naturally within the erythroid cell, isolated ri-bosomal RNAwas most susceptible to the action of theenzyme, intact ribosomes least susceptible, and transferRNA intermediate between them. Natural substrateswere degraded completely to nucleotides in cell lysates.Competitive inhibition studies indicate that one enzymesystem is capable of degrading both RNA and ribo-somes, although the existence of more than one enzymehas not been excluded. Erythroid cell lysates quicklybroke down polyribosomes into single ribosomes. Themore rapid degradation of ribosomes, as compared withtransfer RNA, which occurs in vivo, as opposed to find-ings in vitro, suggests that there is a special intracellu-lar mechanism responsible for ribosome degradation inthe maturing erythroid cell.

INTRODUCTIONMaturation of the mammalian erythroid element is ac-companied by a decrease in RNA content of the cell(1). Degradation is the sole means of altering the cellcontent of RNAin the late stages of erythroid cell matu-ration, because the mammalian reticulocyte lacks the ca-pacity to synthesize RNA (2). The maturing erythroidcell, therefore, offers a unique opportunity to study therelationship between cell maturation and degradation ofRNA. Farkas and Marks have demonstrated that an

Received for publication 21 April 1969 and in revised form26 May 1969.

RNase extracted from the soluble fraction of rabbitreticulocytes is capable of degrading a natural substrate,reticulocyte RNA, and of attacking reticulocyte poly-ribosomes (3). Other attempts to characterize theRNase of erythroid cells (4-10) have given contradic-tory reports as concerns pH optima, substrate specificity,inhibitory substances, and location within the cell.Thus far, it has not been conclusively demonstrated thatany of the isolated enzymes are responsible for degra-dation within the cell.

Clear understanding of the relationship between RNAdegradation and erythroid cell maturation requiresknowledge of events as they occur within the cell. Aprevious report has outlined features of RNAdegrada-tion in intact erythroid cells (11). The present study,which utilizes an in vitro erythroid cell lysate systemwhich approximates the characteristics of degradationof natural substrates in vivo, further defines aspects oferythroid RNAdegradation as it occurs in a physiologicenvironment. The characteristics of the process indicatethat the RNase isolated by Farkas and Marks (3) isresponsible for RNA degradation in the erythroid cell.Natural substrates have been found to differ in theirsusceptibility to degradation. In contrast to the findingin the intact cell that ribosomes are more susceptible to

degradation than is transfer RNA (11), in the lysatesystem ribosomes are more resistant to degradation thanin transfer RNA. This suggests that a specific mecha-nism for ribosome degradation must be present in theintact cell.

METHODSIsolation of cells and preparation of cell lysates. Periph-

eral venous blood was obtained from normal New Zealandwhite rabbits or from animals with a reticulocytosis inducedby four daily subcutaneous injections of a 2.5%o solution ofphenylhydrazine hydrochloride (12). The blood was collectedin heparinized syringes 3 days after the final injection and.immediately placed in an ice bath. All subsequent procedureswere done at 0'-4'C. The cells were separated by centrifu-gation and washed twice with 10 volumes of 310 milliosmolarphosphate buffer, pH 7.4. After each wash the buffy coat wasremoved by aspiration. Cells from which ribosomes were to

1724 The Journal of Clinical Investigation Volume 48 1969

be prepared were washed in 0.9% sodium chloride solutionwhich contained 1.5 X 10' M magnesium chloride.

It was necessary to ensure that the cells were not con-taminated with a ribonuclease present in blood plasma.After separation of the red cells by centrifugation ribo-nuclease activity was determined in the plasma and in thebuffer used to subsequently wash the cells. In an animal witha hematocrit of approximately 20%o at the time of cellcollection the total nuclease activity in the plasma wasroughly twice that found in the sedimented cells. Packedcells obtained in this manner are contaminated to an extentof less than 3% with plasma (13). The supernatant fluidfollowing the second wash contained only a trace of nucleaseactivity, and none was present in the supernatant from thethird wash. Therefore, twice-washed cells were consideredto be free of exogenous nuclease and were routinely used inthe assay system to be described.

Cells were lysed by addition of four volumes of hypotonicsolution, either 7.37 X 10' m sodium phosphate buffer, pH7.4, distilled water, or a solution of 1.5 X 10' M magnesiumchloride in 1 X 10' M Tris, pH 7.5 (solution A). Membrane-free hemolysates were prepared by centrifugation at 17,300 gfor 10 min.

Preparation of ribosomes and RNA. Erythroid cell RNAand RNA-'P were prepared and purified by the phenolmethod as previously described (14). After dialysis for18 hr at 4VC the RNAwas stored at -200C in 0.1 M sodiumacetate buffer, pH 5.1. Ribosomes were prepared by ultra-centrifugation of membrane-free hemolysates at 226,000 gfor 90 min. Ribosomes were stored in solution A at -20'Cuntil used, or ribosomal RNA was prepared from them.Soluble RNAwas prepared from the 226,000 g supernatant.This fraction has previously been shown to be free of sig-nificant contamination with ribosomal RNA (15). Sucrosedensity gradient centrifugation was carried out as previouslydescribed (12).

Analytical methods. Hemocytometry, ennumeration ofreticulocytes, and determination of hematocrit were done bystandard methods. RNA in erythroid cells was extracted anddetermined as previously described (16). Nonerythroid ele-ments of the blood have been shown not to significantlyaffect the results of these determinations (16).

Assay of ribonuclease activity. Ribonuclease activity wasdetermined at 37° in a 1 or 2 ml system consisting of wholehemolysate or membrane-free hemolysate, 7.37 X 10' Mphosphate buffer, and an appropriate 'P-labeled substrate.The substrates used were total RNA, ribosomal RNA,transfer RNA, or ribosomes isolated from rabbit reticulo-cytes and added in amounts closely approximating the actualconcentration of RNAin reticulocytes (15). At the initiationand conclusion of incubation ice-cold trichloracetic acid(TCA) was added to a final concentration of 5% in dupli-cate assay tubes. The precipitated, acid-insoluble RNA andprotein were sedimented by centrifugation at 17,000 g for10 min. An aliquot of the clear supernatant, containing acid-soluble RNAbreakdown products, was placed in a countingvial for determination of radioactivity. When additions to theassay system were made, appropriate controls were obtainedat the initiation and conclusion of incubation by adjustingthe amounts of reagents and additives in all tubes to identicalconcentrations. During 4 hr in phosphate buffer at tempera-tures up to 900C native RNA-8P did not break down intoacid-soluble fragments.

Determination radioactivity. 'P radioactivity in TCAextracts, plasma, hemolysates, and substrate material wasdetermined by pipetting 0.2-0.5 ml of the solution directlyinto 10 ml of Bray's solution (17). The vials were counted

30_

U 20_- -

X10_V)

0.5 1.0 1.5 2.0LYSATE (ml)

FIGURE 1 The relationship between the rate of RNAdegra-dation and the amount of whole lysate in the assay system.The substrate used was 0.15 mg/ml of total reticulocyteRNA-8P in a 2.0 ml system containing appropriate amountsof 7.37 X 10' M phosphate buffer, pH 7.4, and whole lysateprepared in the same buffer. Destruction of RNA during a2 hr incubation at 370C is reported as per cent of totalsubstrate degraded.

in a Packard liquid scintillation counter with an efficiencyof more than 95%o. In all cases sufficient counts were accu-mulated to reduce counting error to less than 3%.

Materials. Rabbits were obtained from commercialsources. All chemicals used were reagent grade. Liquifiedphenol and bentonite were obtained from the Fisher Scien-tific Company, King of Prussia, Pa. Bentonite was preparedas described by Fraenkel-Conrat, Singer, and Tsugita (18).Carrier-free 8P was obtained from Tracerlab Div., Labora-tory for Electronics, Inc.

RESULTS

RNA degradation in whole cell lysates. The initialstudies determined if ribonuclease activity was presentin cell lysates, and if so, whether it resembled that pres-ent in intact cells. The characteristics of RNAdegrada-tion in whole cell lysates, determined by assaying RNAat the beginning and conclusion of a 2 or 4 hr incubationat 370C was compared with that previously observedduring incubation of whole cells (11). The rate of totalRNA degradation in six studies in the intact cell was3.9 +2.5%/hr (11), comparable with the rate observedin the whole cell lysates, 3.4 ±1.7%/hr. Thus, RNAca-tabolism does proceed in whole cell lysates at a rate simi-lar to that in the intact cell. Other similarities betweenRNAcatablism in the intact cell and in whole cell lysates,documented below, included absence of RNAdegradationat 0WC, a temperature optimum of 50'C, and the lack ofsignificant inhibition of RNA degradation by fluoride,cyanide, or iodoacetate.

RNAdegradation in the assay system was proportionalto the amount of whole lysate added to the system overa wide range (Fig. 1). The rate of degradation of RNAby whole cell lysates at 37°C was linear for the initial30 min of incubation, and thereafter decreased (Fig. 2).

Erythroid Cell RNase Activity 1725

aw

w-Jw0.

N%M~

C-

K1001-

onL_>-

60-J

x

:

x

A

20 40 60 80MINUTES 37*

100 120

FIGURE 2 The rate of degradation of reticulocyte RNAbywhole lysate during incubation at 370C. Acid-soluble degra-dation products of total reticulocyte RNA-82P were deter-mined at intervals in aliquots of the assay system describedfor Fig. 1.

RNase activity was present in hemolysates prepared fromblood with a high proportion of reticulocytes and fromnormal blood. Activity in normal blood, however, waslower, per unit of hemoglobin, than in blood with areticulocytosis. RNase activity was present in whole celllysates and in membrane-free hemolysates.- Removal ofthe cell membranes by centrifugation did not significantlyalter the characteristics of RNase activity in the hemoly-sates.

pH and temperature optima. The pH optimum forthe rate of RNAdegradation was 7.0 in both phosphateand Tris buffer and fell off sharply as the pH was in-creased (Fig. 3). The stability of RNase activity in thelysates (see below) did not differ over the pH rangetested during a 2 hr incubation.' The effect of tempera-ture on RNA degradation in whole lysates was similarto that observed in the intact cell (11), as shown inFig. 4. No degradation was observed at 0°C and the rateof degradation was maximal at 50°C, although assayswere usually carried out at 37°C. Degradation wass notcompletely abolished at temperatures of 900C.

Requirement for energy. Since the degradation ofRNAin whole lysates did not occur at 0°C investigationswere carried out to determine if energy was requiredfor the reaction. The addition to the assay system ofan energy generating system consisting of final concen-trations of 1 X 10' M adenosine triphosphate (ATP),2 X 10-' M guanosine triphosphate (GTP) 5 X 10' Mphosphoenol pyruvate, and 100 yg/ml of pyruvate kinase,did not enhance RNase activity. Inhibitors of eitheranerobic glycolysis, iodoacetate or fluoride, or oxidativeglycolysis, cyanide, at concentration of 10 moles/liter

1Burka, E. R. Unpublished observations.

Fr

40F

20 _

I I I l I I I

5.0 6.0 7.0 8.0 9.0pH

FIGURE 3 The relation of RNase activity in whole lysatesto pH. The lysates were prepared in 7.37 X 10' M phosphatebuffer of the indicated pH and assayed for RNase activityunder the conditions described for Fig. 1. A similar curvewas noted in lysates prepared with 1 x 10' M Tris buffer.

had only a minor effect on enzyme activity (Table I).These studies indicate that the degradation of RNAbyerythroid cell RNase is not an energy-dependent process.

Requirement for ions. Lysates used in the assay sys-tem were routinely made in 7.37 X 10' M sodium phos-phate buffer. As shown in Fig. 5, this buffer did notinhibit enzyme activity in concentrations up to 50mmoles/liter. RNase activity did not have an absoluterequirement for inorganic ions. Cell 1vsates made with

100 _

>- 80\

4 60 -

xS 40_

20-

0 20 40 60 80 100DEGREESCENTIGRADE

FIGURE 4 The relationship between RNase activity in wholelysates and temperature. Assay of whole lysates was carriedout as described for Fig. 1 for 1 hr at the indicated tem-peratures using 0.19 mg/ml total RNA-'P as a substrate.

1726 E. R. Burka

distilled water, or dialyzed for 18 hr against 1 X 10' Methylenediaminetetraacetate (EDTA), pH 7.5, had ac-tivity between 82 and 112% of that in lysates made inphosphate buffer. Addition of either magnesium chlorideor calcium chloride to the assay system caused a con-centration-dependent loss of enzyme activity. This effectwas due to the cations, as addition of similar concentra-tions of sodium chloride did not inhibit enzyme activity.Salt concentrations of 0.5 mole/liter abolished enzymeactivity.

Effect of modifiers. The effects of known modifiers ofenzyme activity are shown in Table I. Sodium deoxy-cholate was a strong inhibitor of RNase activity inwhole cell lysates. The enzyme, like other ribonucleases,was adsorbed by bentonite. Addition and subsequent re-moval by centrifugation of either 4 or 12 mg of ben-tonite/10 ml of whole lysate caused a decrease in RNAdegradation to 78 and 11%, respectively, of control ac-tivity. The addition of bentonite directly to the assaysystem also resulted in a loss of enzyme activity. Thepresence of n-ethylmaleimide or p-chloromercuribenzoate,agents which block sulfhydryl groups, was not inhibitory

TABLE IEffect of Inhibitors on Whole Reticulocyte

Lysate RNase Activity

ActivityInhibitor Concentration remaining

Bentonite* 0.4 mg/ml 781.2 mg/ml 11

Deoxycholate 0.1% 50.25% 2

Fluoride 5 X 10-3M 911 X1O-,2M 91

Cyanide 5 X 103-M 1001 X 10-2M 83

lodoacetate 5 X 10-3M 941 X 10 2M 88

n-Ethylmaleimide 1 X 10-6M 1061 X 10 M 1022 X 10-4M 82

p-Chloromercuribenzoate 1 X 10-6 M 1081 <10- M 982X10--4 -100

RNase activity in whole iysates was determined as describedfor Fig. 1 in the presence of the indicated substances. Degreeof inhibition is expressed as the percent of enzymatic activityin the treated iysates as compared to that observed in a con-trol assay.* Added to whole lysates and then removed by centrifugationbefore assay for RNase activity.

* 'SL* ~~~~O.vMgF 50F

^ ~~~~~~Ca25L

1. I I I I I

0 10 20 30 40 50CONCENTRATION(mM)

FIGURE 5 The effect of ionic strength on RNase activity inwhole lysates. The lysates were prepared with distilledwater and appropriate amounts of 1 M Na2HPO, CaClk, orMgCIa added to bring the system to the indicated molaritybefore assay of enzyme activity against a concentration of0.08 mg/ml of total RNA-"P as described for Fig. 1.Results are expressed as per cent of degradation observedin the absence of the indicated ions.

at concentrations of less than 10-' moles/liter. At greaterconcentrations n-ethylmaleimide caused a slight inhibi-tion of enzyme activity. As might be expected from therelative lack of effect with these agents, the additionof reducing agents such as dithiothreitol or mercapto-ethanol did not increase or prolong enzyme activity.

Stability of RNase activity. During incubation ofwhole lysates at 370C RNase activity was progressivelylost. The rate of loss was initially rapid, but decreasedwith the time of incubation. An average of 34% (range14 44) of the initial activity remained after 22 hr at

F

-J

4R

501-

25F

I I I I I5 10 15 20 25

HOURS37°FIGuRE 6 The decline in RNase activity in whole lysatesduring incubation at 370C. At intervals aliquots of theincubated lysate were assayed against a concentration of0.19 mg/ml of total RNA-"P as described for Fig. 1. Theshaded area represents the range of values observed in fourseparate studies.

Erythroid CeU RNase Activity 1727

75

a _t/o - F''M -

W400 - / sr-

O O

W4IQIQ I I I I I

5 10 15 20 25 30 35RIBOSOMALRNA (Mx10-8)

FIGURE, 7 The relationship between degradation of RNA by wholelysates and substrate concentration. Assays of whole lysates were carriedout over a 30 min period to insure linearity of the rate of degradation.The inset shows a double reciprocal plot (Lineweaver-Burk) of thedata, giving a K. of 12.5 X 10 ' moles/liter for ribosomal RNA. Themolecular weight of rRNA was assumed to be -2 X 10' (15).

370C (Fig. 6). At - 20'C approximately 80% of ac-tivity remained after 2 days of storage, and 61% of theactivity was still present on the 15th day of storage.

Kinetics of RNA degradation by whole cell lysates.Fig. 7 shows the relationship between the concentrationof substrate and the rate of RNAdegradation by wholecell lysates. Incubations in these studies were carriedout for only 30 min to insure linear rates of substratedegradation (see Fig. 2). The rate of degradationasymptotically approaches a maximum as the substrateconcentration is increased. A Lineweaver-Burk plot ofthe data is shown in the inset in Fig. 7. The double re-ciprocal plot shows that RNA degradation in wholecell lysates follows Michaelis-Menten kinetics. The cal-culated Km for the substrate used, reticulocyte ribosomalRNA, is 12.5 X 10' moles/liter. These data, along withthe finding that RNAdestruction is proportional to theamount of lysate in the assay system (Fig. 1), and thecharacteristic effect of temperature (11) strongly sug-gest that an enzyme is responsible for destruction ofRNA in whole lysates.

Susceptibility of substrates. The susceptibility of thenatural substrates, ribosomal RNA, transfer RNA, andribosomes to degradation by whole lysates was not simi-lar. Degradation of all substrates followed Michaelis-Menten kinetics, and the K. for each of these substrateswas determined as illustrated for ribosomal RNA in

Fig. 7 (Table II). To further investigate the suscepti-bility of the various substrates to enzyme action, ap-proximately equal amounts of the four substrates wereplaced in assay systems and at intervals during a 2 hrincubation aliquots were removed for determination ofacid-soluble fragments of RNA (Fig. 8). RibosomalRNAwas most susceptible to the action of the enzyme.Total RNA, prepared from rabbits with a reticulo-cytosis of more than 90%, and therefore consisting of

TABLE I ISubstrate Kinetics of Whole Lysate RNase

Rate of substratedegradation, per

cent per hourat 370C

Km N* Per cent Range

Total RNA 16.7 X 10-8 M 3 21 20-21Ribosomal RNA 12.5 X 10-8 M 6 23 17-32Transfer RNA 6.1 X 10-6 M 5 9.8 6-14Ribosomes 2.0 X 10-6 M 4 5.5 1-11

Km of reticulocyte RNA-'2P substrates was determined asillustrated in Fig. 7. Rate of substrate degradation was deter-mined during the 1st hour of incubation of studies as illustratedin Fig. 8.* Number of determinations.

1728 E. R. Burka

more than 75% ribosomal RNA (15), was destroyedat a rate only slightly less than that of isolated ribosomalRNA. Transfer RNA was destroyed at approximatelyhalf the rate of that of ribosomal RNA. Intact ribo-somes were relatively resistent to degradation by eryth-roid cell RNase, only 2% of the labeled rRNA in theparticles becoming acid soluble over a 2 hr period. Theserelative degrees of susceptibility were consistent, asshown by the average rates of destruction of varioussubstrates in several studies (Table II). These resultsindicate that although isolated ribosomal RNA is quitesusceptible to the action of the enzyme, in the form inwhich it naturally occurs within the cell, organized withprotein in ribosomes, it is quite resistent to enzymaticdegradation. Deoxyribonucleic acid (DNA) was notbroken down upon incubation with erythroid cell lysates.

Although detailed analysis of the products of degrada-tion were not carried out, an estimation of the com-pleteness of RNA degradation in erythroid cell lysateswas obtained by comparing the relative amounts ofproduct soluble in 5% TCA and in uranium salts inTCA. Solutions of uranyl acetate in TCA quantitativelyprecipitate all compounds larger than nucleotides (19),while some short-chain oligonucleotides, probably con-taining less than four nucleotides, remain soluble in5% TCA (20). Duplicate assays of RNase activitywere carried out with various substrates and precipita-tion was done with a final concentration of either 5%TCA or 5% TCA containing 0.25% uranyl acetate. Theproportion of the final degradation products soluble inTCA but insoluble in uranyl acetate, representing oligo-nucleotides, averaged 6.2 ±1.4% for ribosomal RNA,26.6 +2.5% for transfer RNA, and 52.3 +17.6% forribosomes. Although this test system does not givedetailed data on the size of oligonucleotide fragments,the almost total solubility of degradation products ofisolated ribosomal RNAin uranyl acetate indicates thaterythroid cell lysates are capable of degrading RNAcompletely to nucleotides. The lesser degree of completedegradation observed with transfer RNA or ribosomesprobably reflects the differences in the rates of degrada-tion of these substrates (see above), and suggests thatenzymatic degradation of RNA in the erythroid cellproceeds by steps through a stage of oligonucleotidefragments. It has not been definitely ruled out by thesestudies that small degration products of ribosomes arecoprecipitated with protein by the acid reagents. Thisappears unlikely, however, since transfer RNA wasalso not fully degraded and almost half of the total ribo-somal degradation did proceed fully to nucleotides.

These studies indicated that substances were presentin erythroid cell lysates which degraded both nativeRNAand ribosomes. To determine whether one enzymewas capable of destroying both RNAand ribosomes, or

15 30 60 120MINUTES INCUBATION

FIGURE 8 Comparison of the rates of degradation ofnatural substrates by whole lysates. Approximately equalconcentrations of substrates were used: ribosomal RNA,0.13 mg/ml; total RNA, 0.15 mg/ml; and transfer RNA,0.18 mg/ml. The concentration of ribosomes used, 0.3 mg/ml,contains 0.15 mg/ml of ribosomal RNA.

whether more than one enzyme might be involved,studies were carried out to see if there was competitiveinhibition between the substrates in question. RNaseactivity in cell lysates was assayed against increasingconcentrations of rRNA-82P both in presence and ab-sence of a constant amount of unlabeled ribosomes.The results are shown as Lineweaver-Burk plots inFig. 9. The Km with rRNA as a substrate, 11.1 X 10'moles/liter, is in close agreement with that of theseparate study shown in Fig. 7. In the presence of ribo-somes the intercept on the ordinate is unchanged, but theKm is increased; these findings indicate that there iscompetitive inhibition between the two substrates. Thiswas confirmed when the study was done using labeledribosomes as the substrate and unlabeled ribosomal RNAas the inhibitor. Although this indicates that one enzymesystem is capable of degrading both native RNA andribosomes within the erythroid cell, this does not provethat more than one enzyme might not be present.

Effect on ribosomes. Since the bulk of erythroid cellRNAoccurs naturally as ribosomes (15) further inves-tigation of the effect of erythroid cell RNase on reticulo-cyte ribosomes was undertaken. Reticulocyte ribosomeshave been reported to contain an RNase (6, 7) but thisfinding has been disputed (8). It was, therefore, firstnecessary to determine if significant autodegradation ofribosomes occurred during incubation at 37°C. During a6 hr incubation at 37°C in solution A less than 0.6% of'P-labeled reticulocyte ribosomes were degraded toacid-soluble products. In contrast, ribosomes incubated in

Erythroid Cell RNase Activity 1729

the presence of whole lysate prepared with the samebuffer degraded 7.7% of their RNA into acid-solublefragments. This slow rate of degradation was not, how-ever, the only action of erythroid cell RNase on ribo-somes. Fig. 10 illustrates that during incubation ofreticulocyte ribosomes with whole cell lysates for 30 minthere was virtually complete breakdown of polyribosomesto single ribosomes. The breakdown of polyribosomesoccurred after as little as 10 min incubation. No loss ofribosomal RNA-82P was detected in this short period.Breakdown of polyribosomes incubated under the sameconditions in solution A did not occur, and, in fact, wasonly minimal after 7 hr of incubation.' It is unlikely thatthe observed disappearance of polyribosomes was due tocontinuing protein synthesis, since in the absence ofenergy protein synthesis does not occur (21). Thesedata indicate that reticulocyte ribosomes do not containan RNase capable of causing either significant auto-degradation or breakdown of polyribosomes, whereas inwhole cell lysates there is an enzyme which causes aslow degradation of ribosomes and a rapid conversion ofpolyribosomes to single ribosomes.

DISCUSSIONThe kinetics of degradation of RNA in erythroid celllysates indicate that RNAdestruction within the mam-malian erythroid element is an enzyme-mediated process.Although the lysate system used for the present studiesdoes not provide detailed information relating to anisolated enzyme, the study of RNA degradation underthe conditions described demonstrate characteristics ofthe process applicable to actual in vivo conditions, asopposed to artificial laboratory situations. The charac-

3

'v 2

+ RIBOSOMES

-0.05 ~' 0.05 alo 0.15 0.20!,-s x 10- 9

FIGURE 9 Competitive inhibition of degradation of ribo-somal RNA by ribosomes. Assays of RNase activity inwhole lysates were carried out as described for Fig. 7 inthe presence of 1 mg/ml of unlabeled ribosomes with vary-ing amounts of rRNA-'P as the substrate.

14

10

1 1

C)

(, 6

8m4

0.

M0-0

2

1 5 10*- TUBE NUMBER

15

FIGURE 10 The effect of whole lysate on ribosome structure.Free 'P-labeled reticulocyte ribosomes were incubated for30 min in either solution A ( 0 ) or whole lysate prepared insolution A (0). Equal amounts of the incubation suspensionswere layered over an 11.4 ml 10-30%o linear gradient ofsucrose in solution A and centrifuged at 40C for 1 hr at40,000 rpm in the Spinco SW40 rotor. Eight-drop sampleswere collected directly into vials containing 10 ml of scintil-lator and counted to determine distribution of ribosomes-82Pin the gradient. The direction of sedimentation is from rightto left. Tube 10 represents a sedimentation coefficient of 80S.

teristics of this process strongly suggest that thereticulocyte RNase isolated by Farkas and Marks (3)is responsible for RNAdegradation in the erythroid cell.Properties of the enzyme which they isolated fromreticulocyte hemolysates which are similar to those ofdegradation in the lysate system include pH optimum,the effect of temperature on activity, requirements forand inhibition by ions, ability to break down polyribo-somes, and relative insensitivity of enzyme activity tothe effects of sulfhydryl donors and inhibitors. This lastfact makes it unlikely that oxidation of the enzyme isrelated to the loss of activity in cell lysates at physio-logic temperatures.

Certain differences between the studies in isolatedsystems and the present ones emphasize the importanceof physiologically oriented investigations. Hemoglobin,the major intracellular constituent of the erythroid cell,has been reported to inhibit the action of an RNasepurified from erythroid cells in concentrations of 1.2mg/ml (3). In the present studies erythroid cell RNasewas active against physiologic concentrations of reticulo-cyte RNAor ribosomes in the presence of about 50 mg/ml of hemoglobin. Thus, inhibition by hemoglobin is oflittle physiologic importance. Heme also inhibits both

1730 E. R. Burka

purified erythroid cell RNase (3) and RNase activityin reticulocyte lysates (22). It is unlikely, however, thatsignificant intracellular concentrations of free heme arepresent in vivo (23).

The evidence, on the basis of solubility of the productin dilute acid solutions of uranyl acetate (19), thatdegradation of isolated RNA in erythroid cell lysatesproceeds fully to nucleotides is consistent with thestudies of Bertles and Beck (24). They demonstratedthat in the maturing reticulocyte, RNA is completelydegraded to low molecular weight products which aremetabolized intracellularly or diffuse out of the cell.The partially purified enzyme of Farkas and Marksshowed the major product of degradation of liver ribo-somal RNA to be acid-soluble oligonucleotides with anaverage chain length of 6 bases and having a 3' phosphateterminus (3). If, as the present studies suggest, thisendonuclease is responsible for RNA degradation inthe intact erythroid cell, phosphodiesterases must bepresent which further carry the breakdown to comple-tion. The observation that degradation of transfer RNAand ribosomes in erythroid cell lysates proceeds througha stage of short-chain oligonucleotides supports thisinterpretation. Detailed data concerning phosphodiester-ase or monoesterase activity in rabbit erythroid cells isnot available.

Farkas and Marks noted that their isolated enzymedegraded transfer RNAat a rate roughly twice that ofribosomal RNA (3). This is in contrast to the resultsreported here that the reticulocyte lysate system degradesribosomal RNAmore rapidly than transfer RNA. Thesecontradictory results may be due to the use of a differentsubstrate, Escherichia coli transfer RNA, rather thanreticulocyte RNA, with the isolated RNase. Despite therapid rate of degradation of isolated ribosomal RNA inthe lysate system, ribosomes were degraded at a rela-tively slow rate. As the competitive inhibition betweenribosomes and rRNA suggests that one enzyme is capa-ble of degrading both substrates, the organization ofrRNA in ribosomes must be of importance in deter-mining the susceptibility of the substrate to degrada-tion. Despite the relative resistance of ribosomes toenzymatic degradation, there appears to be a ribosomalRNAcomponent, essential for maintenance of polyribo-some structure, which is easily accessible to enzymeaction. Whole erythroid cell lysates quickly caused dis-aggregation of polyribosomes, an action similar to thatof minute amounts of pancreatic ribonuclease (21) andRNases extracted from erythroid cells (3, 5). Thebreakdown of polyribosomes to single ribosomes in thepresence of cell lysates occurs so rapidly that it suggeststhat intracellular mechanisms may be present which, by

balancing RNase activity, maintain a concentration ofpolyribosomes sufficient to support necessary proteinsynthesis. An inhibitor of ribonuclease has been isolatedfrom rabbit reticulocytes (25).

The finding that the rate of degradation of ribosomesin the lysate system was less than that of soluble RNAwas unexpected, since during in vivo maturation of in-tact erythroid cells ribosomes disappear at a rate greaterthan that of soluble RNA (11). The discrepancy betweenthe relative rates of degradation of ribosomes and solu-ble RNA in vitro and in vivo suggests that there is aspecific mechanism within the cell which regulates therate of ribosome degradation. One such mechanism to beconsidered is that ribosomes themselves contain anRNase, but the present studies confirmed the absenceof significant RNase activity in ribosomes (8). Therelatively rapid degradation of reticulocyte ribosomesin vivo, in comparison with soluble RNA, thus cannotbe ascribed to an inherent ribosomal RNase. Althoughthe significance of binding of ribosomes to cell mem-branes remains unknown, it is possible that this playsa part in ribosome degradation. Two studies have sug-gested that the major portion of erythroid cell RNaseis confined to the cell membrane (4, 5), and the eryth-roid cell membrane is, in itself, capable of degradingendogenous RNA (11).

The nature of the association between decreasingRNA content and erythroid cell maturation remainsobscure. High concentrations of magnesium have beenreported to retard reticulocyte maturation (26). Theobservation that magnesium ions inhibit erythroid cellRNase in cell lysates suggests, but does not prove, thepossibility that RNase activity is involved in cell matu-ration. Reticulocyte maturation has also been reportedto be slowed by inhibitors of glycolysis (27). The pres-ent work, however, showed no effect by inhibitors ofglycolysis on RNase activity in erythroid cell lysates.Further studies of erythroid RNA degradation in asystem oriented toward physiologic conditions, presentlyin progress, may clarify this relationship, and furtherelucidate the role of the cell membrane in erythroidcell RNA metabolism.

ACKNOWLEDGMENTThis investigation was supported by U. S. Public HealthService Grants HE-10473 and H-6374.

REFERENCES1. Grasso, J. A., J. W. Woodard, and H. Swift. 1963.

Cytochemical studies of nucleic acids and proteins inerythrocytic development. Proc. Nat. Acad. Sci. U. S. A.50: 134.

2. Marks, P. A., E. R. Burka, and D. Schlessinger. 1962.Protein synthesis in erythroid cells. I. Reticulocyte ribo-

Erythroid CeU RNase Activity 1731

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