On ribonuclease activity in reticulocyte ribosomes

6o6

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 95124

ON RIBONUCLEASE ACTIVITY IN RETICULOCYTE RIBOSOMES L. S T A V Y , M. F E L D M A N

AND D. E L S O N

The Weizmann institute o/ Science, Rehovoth (Israel) (Received J u n e i 7 t h , 1964)

SUMMARY

RNAase (polyribonucleotide 2-oligonucleotidotransferase (eyclizing), EC 2.7.7.16) activity of reticulocyte ribosomes of rabbits was tested in comparison to the nucleolytic activity of liver ribosomes. Reticulocyte ribosomes showed practically no RNAase activity under conditions where such activity was demonstrated in liver ribosomes.

INTRODUCTION

It is generally accepted that the amino acid sequence of a specific protein molecule is determined by the nucleotide sequence of a specific molecule of template or messenger RNA. This messenger RNA is believed to be a complementary copy of a specific segment of DNA and to attach itself to the ribosomes, where it directs the synthesis of the protein. The messenger RNA of certain bacteria turns over rapidly 1, and a similar phenomenon appears to have been observed in mammalian cells2. The reticulocyte cell, although capable of prolonged protein synthesis, contains no DNA and should be unable to synthesize new messenger RNA. Thus, it has been generally believed that the reticulocyte must contain a stable messenger RNA, and, indeed, there exists evidence which indicates that this cell does not contain a rapidly renewed RNA (ref. 3). Ribosomes of Escherichia coli and many other organisms contain ribonuclease (RNAase or polyribonucleotide 2-oligonucleotidotransferase (cyclizing), EC 2.7. 7.16) 4. It has been suggested that tile function of this ribosomal enzyme might be to detach messenger RNA from the ribosomes and perhaps to inactivate it ~, although other enzymes probably effect its final degradation 6& If this is indeed the function of the ribosomal RNAase, then reticulocyte ribosomes might be expected to contain relatively less of this enzyme, and perhaps none. The literature contains little definite information on this point. We have therefore compared the RNAase activity of reticulocyte ribosomes with that of ribosomes from an organ which presumably contains a metabolically labile messenger RNA, namely, liver. Our results show the reticulocyte ribosomes to exhibit virtually no RNAase activity under conditions where such activity is easily demonstrated in liver ribosomes. MATERIALS AND METHODS

Rabbit reticulocytes were obtained by intramuscular injections of phenylhydrazine 8. The reticulocytes were isolated and lysed according to LAMFROM 9, with a modification Biochim. Biophys. Acta, 9I (1964) 6 o 6 - 6 i i

RNAAsE

OF RETICULOCYTE RIBOSOMES

607

aimed at getting a relatively pure population of reticulocytes. Following each centrifugation of cells, the layers containing mostly reticulocytes (as determined b y cell counts) were transferred to other tubes, and then further washed. Reticulocyte ribosomes were prepared b y the procedure of EDELMAN et al. TM. The medium employed was o.14 N[ KC1, o.ooi M MgCI~, o.oi IV[Tris-HC1 buffer (pH 7.4). The ribosomes were washed b y three cycles of low (18 ooo ×g, io rain) and high (lO5 ooo ×g, 2 h) speed centrifngation. Liver microsomes, isolated according to the procedure of L I T T L E F I E L D AND K E L L E R 11 a n d K E L L E R AND ZAMECNIK12, were suspended in 0.05 M KCI, o.o15 iV[ MgCI~, 0.025 iV[ Tris (pH 7.6). Sodium deoxycholate was added to a final concentration of 0.5 %, and the ribosomes were isolated by two further cycles of centrifugation. Ribosomes from both liver and reticulocytes were kept at --15 °, as precipitates. For testing RNAase activity, reticulocyte ribosomes were resuspended in the reticulocyte medium. Liver ribosomes were resuspended in liver medium, with a Tris concentration of 0.005 M. The amount of RNA was determined by ultraviolet absorption after total alkaline hydrolysis. RNAase activity was followed b y observing the amount of R N A degraded to acid soluble fragments on incubation at 37 ° or, in one case (Table VI), at o °. Penicillin (200 units/ml) was sometimes included in the incubation mixture to prevent bacterial contamination, but identical results were obtained without penicillin. To I ml of incubation mixture, or an aliquot diluted to I ml with water, were added 2 ml of cold 0.2 N[ HC104 in 50 % ethanol. After mixing, the suspension was left at room temperature for lO-2O Inin and centrifuged at about I S o o × g for 15 rain. The absorbancies of the supernatants were measured at 260 m/~ and 290 rot,. Results are expressed as the difference between the two absorbancies (A260). RNA was prepared b y phenol extraction of lysed E. coli protoplasts 13. When urea was employed, a fresh solution (British Drug House, analar grade) was made up for each experiment. RESULTS

We have tested reticulocyte ribosomes for RNAase activity over a fairly wide range of p H and ionic strength. Liver ribosomes were tested in parallel in order to provide a comparison. I n early experiments, not reported here, we employed rat-liver ribosomes. The experiments were then repeated in greater detail with rabbit-liver ribosomes, so that the final comparison was made with ribosomes of another organ of the same animal. The rat-liver ribosomes were much more active than the rabbit-liver particles. In both cases a pI-{ optimum of about 8 in phosphate was observed, with an optimum I of o.o5-o.1. Tables I, I I and I n illustrate a typical experiment in which rabbit-liver and reticulocyte ribosomes were examined in parallel. Table I shows the course of self digestion (degradation of the ribosomal RNA b y the ribosomal RNAase) over a period of 18 h. These "digested" ribosomes were then incubated for an additional 6 h at different p}{'s with added purified RNA as substrate (Table II). In Table I I I those data of Table n which were obtained at p H 8 are compared with simultaneously run controls. The liver ribosomes underwent a steady self digestion (Table I) and also hydrolyzed exogenous R N A (Table n ) , clearly demonstrating nucleolytic activity. The reticulocyte ribosomes also showed an increase in acid soluble ultraviolet abBiochim. Biophys. Acta, 91 (1964) 6 o 6 - 6 n

608

L. STAVY, M. FELDMAN, D. ELSON TABLE i RABBIT-LIVER

AND RETICULOCVTE

RIBOSOMES:

SELF

DIGESTION

R i b o s o m e s were incubated at 37' in s o d i u m p h o s p h a t e buffer (pH 7-5, l o.o5) containing penicillin (2oo units/ml). At the specific times o . i - m l samples were w i t h d r a w n , diluted with o.9 iall HzO, and precipitated b y the addition of 2 ml of o.2 M HC104 in 5 ° % ethanol. U n d e r these conditions complete conversion of the ribosomal R N A to acid soluble f r a g m e n t s would h a v e given A2e0 values of o.395 (liver) and o.655 (reticulocytes). Incubation time (h)

Liver

Retieulocytes

o.o41 o. i48 o.165 o.214

o

3 6 18

Acid soluble (,]zs~) as per cent o/ total A26o

Acid soluble (A28o)

Liver

o.o37 o.o42 o.o44 0.059

Reliculocyles

lO. 4 37.5 41.8 54.2

5.6 0.4 6. 7 9.o

TABLE II RABBIT-LIVER AND RETICULOCYTE RIBOSOMES: RNAAsE ACTIVITY AGAINSTEXOGENOUS SUBSTRATE E a c h value was o b t a i n e d from a separate i n c u b a t i o n mixture. E a c h m i x t u r e contained o.i ml of ribosomes p r e i n c u b a t e d for 18 h (taken f r o m the e x p e r i m e n t of Table 1), o.i ml of purified E . coli RNA, 0.25 ml of stock s o d i u m p h o s p h a t e buffer of I 0.2, and 0,55 ml of H,O. The m i x t u r e s were i n c u b a t e d at 37 ° for the indicated times and were precipitated b y the addition of 2 ml of 0.2 M HC10, in 5 ° % ethanol. U n d e r these conditions complete conversion of the added E . coli R N A to acid soluble f r a g m e n t s would h a v e given a Zl260 value of o.436 above the b a c k g r o u n d value of the degraded ribosomal RNA.

Source o] ribosomes

Incubation time (k)

Liver

Reticulocytes

Acid soluble A26o 6

7

p H o/ stock bu//er 8

O 3 6

O.2II 0-257 0.298

-0.306 0.372

0.209 0.343 0.398

O.314 0.351

-0.303 0.327

o 3 6

0.044 0.055 0.057

0.049 0.055 0.063

0.050 0.068 0.066

-0.053 0.069

-0.065 0.063

9

zo

TABLE III RABBIT-LIVER AND RETICULOCY'IE RIBOSOMES: RNAAsE ACTIVITY AT p H 8 AND f 0.05 The conditions and q u a n t i t i e s were the same as those of Table II. The i n c u b a t i o n m i x t u r e s contained ribosomes alone (line I), E . coli R N A alone (line 2), or b o t h t o g e t h e r (line 4). Acid soluble Aa6o Source o] ribosomes Liver

Reticulocytes

o

C o n t r o l - r i b o s o m e s alone C o n t r o l - R N A alone S u m of controls (I plus 2) R i b o s o m e s plus R N A i n c u b a t e d t o g e t h e r Increase over controls (4 m i n u s 3)

3

Incubation time (h) 6 o

3

6

o.214

o.220

o.219

o.o54

0.068

0.064

O.OLO

o.oi 4

o.o16

o.0o 9

o.o12

o.oi 5

0.228 o.2o9 o

0.234 o.343 O.lO9

0.235 0.398 o.163

0.063 0.050 o

0.080 0.068 o

0.079 o.o66 o

Biochim.

Biophys.

Acta,

91 (1964) 6o6-611

RNAAsE OF RETICULOCYTE RIBOSOMES

6O9

sorbing material (Tables I, II). The increase is very small, but it seems too large to represent a reasonable experimental error and we have observed it in a number of experiments. It may, therefore, indicate the presence of traces of RNAase activity in the reticulocyte ribosome preparations. The data of Table III, however, show that the breakdown of added exogenous R N A is not perceptibly accelerated in the presence of reticulocyte ribosomes. Thus, it is not certain that the observed slight degradation of ribosomal ~RNA was due to ~RNAase action. In any event, it is clear that if the reticulocyte ribosomes exhibited RNAase activity in these experiments, the amount of activity was minute. Similar results were obtained under all of the conditions tested: 37 °, pI-I 5-11, I O.Ol-O.2. These results are not to be explained in terms of an unusual stability of the R N A of reticulocyte ribosomes toward nucleases. When reticulocyte ribosomes were incubated with a smaller quantity of rat-liver ribosomes, the reticulocyte R N A was rapidly degraded by the nuclease of the liver particles (Table IV). Neither can TABLE IV RABBIT-RETICULOCYTE RIBOSOMES: DIGESTION BY RAT-LIVER RIBOSOMAL RNAAsE R i b o s o m e s were i n c u b a t e d at 37 ° in s o d i u m p h o s p h a t e buffer (pH 6.8, I 0.05). Samples w e r e t a k e n and acid soluble A2e0 was determined as described in Table I. The same q u a n t i t i e s of ribosomes were i n c u b a t e d separately a n d together. U n d e r these conditions complete conversion of the ribosomal R N A to acid soluble f r a g m e n t s would h a v e given 212e0 values of 0.374 (rat liver), 0.632 (rabbit reticulocytes) and 1.oo6 (both together). Acid soluble zJ2eo Incubation time

o io 20 3° 60 12

Rat liver

Source o] ribosomes Rabbit reticulocyte

Both

0.040 o.142 0.23o 0.258 0.258 o.255

0.034 --0.033 -o.044

0.052 o.218 o.339 0.368 0.420 0.702

min min min min h

TABLE V RABBIT-RETICULOCYTE RIBOSOMES: EFFECT OF DEOXYCHOLATE R a b b i t - r e t i c u l o c y t e ribosomes were p r e p a r e d in the usual w a y t h r o u g h the first s e d i m e n t a t i o n in the p r e p a r a t i v e ultracentrifuge. The ribosomal pellet was suspended and divided into two equal parts. To one p a r t (marked + in t h e . T a b l e ) was added s o d i u m deoxycholate to a final c o n c e n t r a t i o n of o. 5 %. The o t h e r p a r t (marked - - ) was n o t t r e a t e d w i t h deoxycholate. The purification of b o t h p a r t s was t h e n completed in the usual w a y and the two p r e p a r a t i o n s w e r e a d j u s t e d to equal c o n c e n t r a t i o n s of ribosomes. E q u a l aliquots were i n c u b a t e d at 37 ° in s o d i u m p h o s p h a t e buffers of I o.o 5 at the p H ' s shown. S a m p l i n g was as described in Table I. U n d e r these conditions complete conversion of the ribosomal R N A to acid soluble f r a g m e n t s would have given a A2eo value of 0.65 o. Acid soluble A~so Incubation time

pH

(h)

7.0 Deoxycholate

o 6 24

--

o.O2O o.O25 o.o41

7.5 +

o.o21 O.O18 o.o32

8.0

--

O.O16 O.O17 0.o33 Biochim.

+

--

O.OI2 O.O18 0.029 Biophys.

Acta,

O.O24 0.028 0.027

+

O.OI9 0.02 I 0.034

91 (1964) 6o6-611

61o

L. S'rAVY, M. FELDMAN, D. ELSON

the results be attributed to the main difference between the methods of preparation of the two types of ribosome, namely, the employment of deoxycholate only in the case of the liver preparation. When reticulocyte ribosomes were treated with this reagent, they showed no more RNAase activity than those not so treated (Table V). I t is still possible, however, that the reticulocyte ribosomes contain a latent nuclease which is not released under the conditions employed, although these conditions sufficed to release RNAase activity from the liver ribosomes. The experiments illustrated in Table VI, while not disproving this possibility, make it seem rather unlikely. Urea, a reagent which disrupts the structure of the ribosome, has been used to demonstrate latent RNAase activity in ribonucleoproteins 4. Table VI shows that although 6 M urea released active nuclease from liver ribosomes, it had no significant effect of this sort on the reticulocyte particles. TABLE VI RABBIT-LIVER

AND

RETICULOCYTE

RIBOSOMES:

EFFECT

OF

UREA

IN THE

COLD

E a c h v a l u e w a s o b t a i n e d f r o m a s e p a r a t e i n c u b a t i o n m i x t u r e i .o ml i n v o l u m e a n d c o n t a i n i n g s o d i u m p h o s p h a t e buffer (pH 8.0, I 0.05). The t u b e s w e re i n c u b a t e d i n a n ice b a t h a n d a s s a y e d as d e s c r i b e d in T a b l e I I . C o m p l e t e c o n v e r s i o n to a c i d s ol ubl e f r a g m e n t s w o u l d h a v e g i v e n t h e f o l l o w i n g d , , 0 v a l u e s : 0.420 (RNA), 0.972 (liver r i b o s o m e s ) , a n d 0.700 ( r e t i c u l o c y t e r i b o s o m e s ) . T h e r e s u l t s are fro m t w o s e p a r a t e e x p e r i m e n t s , one w i t h l i v e r a n d t h e o t h e r w i t h r e t i c u l o c y t e ribosomes. Acid soluble A2eo

In incubation mixture

.Source ol ribosomes Liver Reticuloeyles

Urea conc. (M)

Incubation time (h) o

RNA RNA Ribosomes Ribosomes Ribosomes + RNA Ribosomes + RNA

o 6 o 6 o 6

24

O.OLO O.Ol 4 o.o16 o.o13 -o.oi 2

o.oIo 0.020 0.020 0.200 o. 1 ~o

o

o.o18 O.OLO 0.020 o.o13 0.020 o.o19

24

o.023 o.o19 o.o19 0.036 o.o E8 0.034

DISCUSSION

Our experiments were designed to compare the RNAase activities of ribosomes from cells with metabolically stable and labile messenger RNA's. The incubation conditions were such, however, that other common nucleases might also have been expected to act if they were present. The incubation buffer was sodium phosphate. 3!g2+ and K + ions, present in the ribosome solutions, were added to the incubation mixtures along with the ribosomes. Thus, the incubation media were favorable not only for RNAase, but also for phosphodiesterase (EC 3.I.4.I ), K+-activated phosphodiesterase 7, and polynucleotide phosphorylase (EC 2.7.7.8)t Our results do not constitute proof that these common nucleases are absent froln the reticulocyte ribosome. We have, in fact, observed a slight, but recurring, degradation of the ribosomal RNA, and FARKAS et al. 14 have recently reported a similar finding. Employing a radioactive polynucleotide, these workers also demonstrated activity against an exogenous substrate, which our less sensitive assay method did not detect. However, the levels of nuclease activity observed were low enough Biochim.

B i o p h y s . A c t a , 91 (1964) 6o6-611

~RNAAsE OF RETICULOCYTE RIBOSOMES

611

to give importance to the question of whether the activity might not be due to contaminating ribosomes from cells other than reticulocytes, since it is difficult to prepare reticulocytes absolutely free of other cell types. What we consider worthy of attention in our work is the comparative aspect, namely, that reticulocyte ribosomes contain very much less nuclease activity than do liver ribosomes. This is consistent with the proposal that there may be a correlation between the level of such activity in the ribosome and the metabolic lability of messenger RNA. This proposed correlation should be tested by a comparative study of other cell types. It should also be borne in mind that we have examined only the purified ribosomes, and our results do not apply to other parts of the reticulocyte cell. This is of some interest, since the reticulocyte loses its RNA during maturation, and this is presumably an enzymatic process. ACKNOWLEDGEMENTS

This investigation was supported in part by grants from the United States National Institutes of Health (GM-o5876, C-6165) and the United States National Science Foundation (GB-II63). REFERENCES 1 C. LEVINTHAL, A. KEYNAN AND A. HIGA, Proc. Natl. Aead. Sci. U.S., 48 (1962) 1631. s K. SCHERRER, H. LATHAM AND J. E. DARNELL, Proc. Natl. Acad. Sci. U.S., 49 (1963) 240. 1D. A. MARKS, C. WILLSON, J. KRUH AND F. GROS, Biochim. Biophys. Res. Commun., 8 (1962) 9. 4 D. ELSON, Bioehim. Biophys. Acta, 36 (1959) 372. 5 IVY.TAL AND D. ELSON, Biochim. Biophys. Acta, 76 (1963) 4 o. 6 M. SEKIGUCHI AND S. S. COHEN, J. Biol. Chem., 238 (1963) 349. 7 1D, F. SPAHR AND D. SCHLESSINGER, J. Biol. Chem., 238 (1963) PC 2251. 8 H. ]~ORSOOK, C. L. DEASY, A. J. HAAGEN-SMITH, G. KEIGHLEY AND P. H. LOWRY, J. Biol. Chem., 196 (I952) 669. H. LAMFROM, ]. Mol. Biol., 3 (1961) 241. a0 1. S. EDELMAN, P. O. P. TS'O AND J. VINOGRAD, Biochim. Biophys. Acta, 43 (196o) 393. 11 j. \ v . LITTLEFIELD AND E. B. KELLER, J. Biol. Chem., 224 (1957) 13. 12 E. B. KELLER AND i°. C. ZAMECNIK, J. Biol. Chem., 221 (1956) 45. a s U. Z. LITTAUER AND H. EISENBERG, Biochim. Biophys. Acta, 32 (1959) 320. 14 W. R. FARKAS, M. SINGER AND P. A. MARKS, Federation Proc., 23 (1964) 23.

Biochim. Biophys. Acta, 9I (1964) 6o6-611