The state of messenger ribonucleic acid and ribosomes in the cytoplasm of ethionine-treated rat liver

305

Biochimica et Biophysica Acta, 383 (1975) 305--315 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98242

THE STATE OF MESSENGER RIBONUCLEIC ACID AND RIBOSOMES IN THE CYTOPLASM OF ETHIONINE-TREATED RAT LIVER*

YAETA ENDO, HIROSHI TOMINAGA and YASUO NATORI

Department of Nutritional Chemistry, Tokushima University, School of Medicine, Tokushima (Japan) (Received September 17th, 1974)

Summary The administration of ethionine to female rats causes breakdown of hepatic polysomes. The state of m R N A and monomeric ribosomes after the polysome dissociation was studied. The mRNA was selectively labeled with [ 1 4 C ] o r o t a t e after a low dose of actinomycin D. Sucrose density gradient centrifugation of Triton X-100-treated cytoplasm revealed an accumulation of heterodisperse radioactive material with very large S values. This material was converted to smaller S values with deoxycholate treatment and was extremely sensitive to mild ribonuclease treatment. Since this material was banded at around 1.43 g/cm 3 in CsC1 gradient centrifugation and contained RNA with a distribution of S values characteristic of polysomal mRNA, this material was identified as mRNA-containing ribonucleoprotein particles. The monomeric ribosomes were shown to be dissociated into subunits in the presence of 0.5 M KC1, indicating that these lacked nascent polypeptide chains. When the animals were recovered from the ethionine treatment by subsequent administration of adenine and methionine, the heterodisperse ribonucleoprotein particles and monomeric ribosomes appeared to be utilized for the reformation of polysomes.

Introduction Ethionine, the ethyl analog of methionine, when injected into female rats, induces a marked inhibition of hepatic RNA [1] and protein [2] synthesis and a rapid accumulation of triglycerides in the liver [3,4]. These effects of ethionine are the consequences of a rapid decrease in the hepatic concentration of

* This c o m m u n i c a t i o n is part of thesis submitted by Y. E n d o to T o k u s h i m a University in partial fulfillment of the requirement for P h . D degree.

306 ATP [4--6]. The main site of the ethionine-induced inhibition of protein synthesis is the polysome [2,7] which undergoes a progressive disaggregation into monomeric ribosomes within a few hours after the administration of the analog [8,9]. All of these effects of ethionine, including the disaggregation of the polysomes, are reversed by the administration of methionine or of ATP precursors such as adenine [10,11]. The initial cause for the disaggregation of polysomes was first t h o u g h t to be due to the unavailability of m R N A because the synthesis of RNA is severely inhibited by ethionine [1]. A subsequent study [12] has shown that such is not the case, since the reformation of liver polysomes in the ethionine-treated animals following the administration of adenine plus methionine was found to occur under conditions in which new RNA synthesis was markedly inhibited in the presence of a large dose of actinomycin D. It has been thus assumed that m R N A following dissociation from the polysomes remains intact in the cytoplasm of the ethionine-treated liver. It is not known, however, in what state the mRNA and ribosome monomers exist after their dissociation from the polysomes. We have recently reported that the administration of ethionine to rats causes a prolongation of the half-life of liver mRNA and proposed that the mRNA, after dissociation from the polysomes, exists n o t in the free form but in the form of a protein complex which is resistant to the attack of nuclease [13]. Here we describe our results, which show that the mRNA, in fact, exists in the form of ribonucleoprotein in the cytoplasm of the ethionine-treated liver. We shall also report evidence that the dissociated ribosomes contain no peptidyl-tRNA. Materials and Methods Female albino rats of Wistar strain weighing approx. 200 g were starved overnight before use. [~4 C]Orotate (20.6 Ci/mol) was obtained from Daiichi Pure Chemicals Co., Tokyo. D L-Ethionine was purchased from Sigma Chemical Co., St. Louis. Actinomycin D was purchased from Mann Research Lab., New York. Treatment o f animals. Actinomycin D(0.55 mg/kg body weight) was injected intraperitoneally into rats 1 h before the injection of 20 pCi of [14 C]orotate. 1 h after the administration of [14 C] orotate, 8 ml of DL-ethionine solution (200 mg) was injected intraperitoneally, and animals were sacrificed 5 h afterwards. For the recovery experiments, the ethionine-treated rats received 10 ml of a solution containing DL-methionine (180 mg) and adenine (20 mg) 1 h before the sacrifice. Preparation of polysomes. Rats were killed by decapitation and livers were quickly excised and homogenized in 3 vol. of ice-cold 50 mM triethanolamine • HC1 buffer (pH 7.6) containing 5 mM MgC12, 25 mM KC1, and 0.25 M sucrose with a Potter-Elvehjem type homogenizer. All isolation procedures were performed at 4 °C. After centrifugation at 12 000 × g for 10 min, the post-mitochondrial supernatant was treated with Triton X-100 (final concentration, 1%) and polysomes were prepared by the m e t h o d of Wettstein et al. [14]. Sucrose and CsC1 gradient centrifugations. Polysomes and purified RNA were analyzed on a linear suCrose gradient as described in the legends to figures.

307 Polysomes and m o n o m e r fractions from sucrose gradients were pooled and centrifuged in CsC1 gradient. After fixing with 6% formaldehyde, CsC1 centrifugation was carried o u t according to the m e t h o d of Brunk and Leick [15] in a Hitachi SW-40 rotor at 38 000 rev./min for 18 h. A b o u t 0.15-ml fractions were collected from the punctured tube. B u o y a n t densities were calculated from the refractive index [16]. Extraction o f R N A from ribonucleoprotein particles. The ribonucleoprotein particles from sucrose gradient were adsorbed on Millipore filters (HA 0.45 pm) and R N A was extracted by the m e t h o d of Infante and Nemer [17]. Radioactivity measurements. For assay of R N A or ribonucleoprotein particles, fractions from sucrose or CsC1 gradients were precipitated with cold 5% trichloroacetic acid. The precipitates were collected on Millipore filters (HA 0.45 pm), washed three times with 15 ml of cold 5% trichloroacetic acid and dried on planchets. The radioactivity was measured by an automatic low-background gasflow counter (Nuclear Chicago, background count of a b o u t 1.4 cpm). For radioactivity measurements in the surcose gradient centrifugation of post-mitochondrial supernatant, fractions were precipitated with cold 5% trichloroacetic acid after the addition of 100 pg casein as carrier. The precipitates were collected on glass fiber filters (Whatman, GF/C), washed, dried and counted in a Packard Tri-Carb liquid scintillation spectrometer with a use of toluene-based scintillator. Results

Accumulation o f heterodisperse radioactive materials in the cytoplasm of ethionine-treated livers In order to study the state of the m R N A released from the polysomes by the ethionine treatment, the m R N A was selectively labeled with [14 C] orotate after the administration of a small dose of actinomycin D (0.55 mg/kg b o d y weight). The rats were then injected with ethionine and the labeling pattern of liver cytoplasm was examined by sucrose gradient centrifugation. At 2 h after the ethionine treatment, the polysome pattern of deoxycholate-treated postmitochondrial supernatant showed slight disaggregation and the radioactive profile essentially followed the absorbance profile of the polysome region (Figs. l a and l b ) . This shows that the labeled m R N A has reached the cytoplasm and integrated into the polysome by 2 h after the ethionine treatment. The sucrose gradient sedimentation pattern of the deoxycholate-treated postmitochondrial supernatant at 5 h after the ethionine treatment is presented in Fig. lc. The polysomes were extensively disaggregated into the m o n o m e r region, and the radioactivity profile followed the absorbance profile. However, when the same post-mitochondrial supernatant was treated with Triton X-100, the absorbance profile was superimposable to that of deoxycholate-treated supernatant, b u t the radioactivity profile was clearly different from that of the deoxycholate-treated sample (Fig. lc). The Triton X-100-treated sample contained heterodisperse radioactive materials with wide range of S values extending over the polysome region. Since the heterodisperse materials appear to be displaced to the materials with smaller S values by the deoxycholate treatment and since the polysome contents appear to be the same in both treatments, the heterodisperse materials do n o t seem to be associated with polysomes.

308

b

C

/ 20OO

2.0

c

E

E c

g

c

1000

1.0

~ ~.o~o ~ . ~ .w ~ ¢:

0

i

10

20

30

]0 Fraction

20 No.

30

]0

2 0/

30 0

Fig. 1. P o l y s o m a l p a t t e r n s a n d r a d i o a c t i v i t y p r o f i l e s a t v a r i o u s t i m e i n t e r v a l s a f t e r i n t r a p e r i t o n e a l inject i o n o f e t h i o n i n e . A n i m a l s w e r e i n j e c t e d w i t h a c t i n o m y c i n D a n d [ 1 4 C ] o r o t a t e as d e s c r i b e d u n d e r Materials and Methods. At various t i m e intervals after injection of ethionine, animals were killed and the p o s t - m i t o c h o n d r i a l s u p e r n a t a n t w a s p r e p a r e d f r o m t h e liver. T h e s u p e r n a t a n t w a s t r e a t e d w i t h e i t h e r s o d i u m d e o x y c h o l a t e ( f i n a l 1%) or T r i t o n X - 1 0 0 (final 1%), a n d a n a l y z e d o n a l i n e a r 1 0 - - 3 0 % s u c r o s e g r a d i e n t in t h e h o m o g e n i z i n g m e d i u m . T h e c e n t r i f u g a t i o n w a s in a H i t a c h i SW-25.1 r o t o r at 25 0 0 0 r e v . / m i n for 4h. - - , A260nm; • ..... - • , r a d i o a c t i v i t y of d e o x y c h o l a t e - t r e a t e d s u p e r n a t a n t ; ( . . . . . . . . ~, r a d i o a c t i v i t y of T r i t o n X - 1 0 0 - t r e a t e d s u p e r n a t a n t , F r a c t i o n s A, B a n d C in (e) r e f e r to the d i f f e r e n t size classes of r a d i o a c t i v e m a t e r i a l s to be a n a l y z e d in Fig. 6. (a) C o n t r o l ( a n i m a l w a s k i l l e d w i t h o u t e t h i o n i n e i n j e c t i o n at 0 t i m e w h i c h w a s 1 h a f t e r i n j e c t i o n o f [ 1 4 C ] o r o t a t e ) . (b) 2 h a f t e r e t h i o n i n e i n j e c t i o n . (e) 5 h after ethionine injection.

The nature of these radioactive materials was then investigated by the Millipore filter binding technique. Most of eukaryotic m R N A s are known to possess a poly(A) sequence and bind to Millipore filters at high ionic strength [ 1 8 , 1 9 ] . As is shown in Table I, about 20--30% of the labeled R N A from the ethionine-treated livers was retained by Millipore filtration. These proportions TABLE I MILLIPORE FILTER BINDING OF RNA FROM ETHIONINE-TREATED

LIVERS

A n i m a l s w e r e i n j e c t e d w i t h a c t i n o m y c i n D a n d [ 14C] o r o t a t e as d e s c r i b e d u n d e r M a t e r i a l s a n d M e t h o d s . At various t i m e intervals after injection of ethionine, animals were killed and p o l y s o m e s (containing h e t e r o d i s p e r s e r a d i o a c t i v e m a t e r i a l s ) w e r e p r e p a r e d . T h e p r e p a r a t i o n o f R N A a n d t h e b i n d i n g of R N A t o M i l i i p o r e filterS w e r e p e r f o r m e d b y t h e p r o c e d u r e o f L e e et ai. [ 1 8 ] . T h e p r o p o r t i o n of t h e r a d i o a c t i v i t y r e t a i n e d o n the f i l t e r r e l a t i v e to t h e t o t a l r a d i o a c t i v i t y o f t h e R N A s a m p l e w a s c a l c u l a t e d . Time after ethionine injection (h)

R N A specific activity (cpm/mg)

Millipore binding (%)

1 2 3 4 C o n t r o l (4 h a f t e r saline i n j e c t i o n )

4960 8540 7 310 8310 25 4 3 0

23.1 24.2 20.7 17.5 27.6

309 were essentially the same as the ethionine-untreated control and remained more or less constant as the polysomes underwent progressive disaggregation by ethionine. This result indicates that, in the ethionine-treated liver, the total mRNA content is unchanged and the bulk of m R N A is not associated with the polysomes. The efficiency of Millipore binding (20--30%) in the present experiment is comparable to the result obtained by Palacios et al. [20] who found that approx. 30--40% of ovalbumin m R N A activity was retained by Millipore filtration under a similar condition.

Utilization of cytoplasmic ribonucleoprotein particles for polysome reformation The administration of adenine and methionine to the ethionine-treated rats brings about a rapid conversion of the monomeric ribosomes to polysomes [ 1 0 , 1 1 , 2 1 ] . For the recovery experiment, the ethionine-treated rats received adenine and methionine intraperitoneally 1 h before the sacrifice. The sedimentation patterns of the ribosomes obtained from the ethionine-treated and the recovered rats are shown in Fig. 2. In the recovered animals the polysomes were reformed and a large portion of the heterodisperse radioactive materials now appeared to be associated with the newly formed polysomes. The supernatant fraction of the sedimented ribosomes was also examined by longer centrifugation in a sucrose gradient for possible occurrence of radioactive peaks at around 40 S or 60 S. There were no definite radioactivities in this region in either ethionine-treated or recovered samples. That the radioactivity was in fact associated with the polysomes in the recovered liver was demonstrated by a ribonuclease digestion experiment. When the ribosomes from the recovered liver were subjected to a very mild ribonuclease treatment, a partial degradation of

Q

A

,~--

Ib

B ----4

I 4.00

1.C

c E

E c o

u~ c

c~

L~

200

0.5

\ 0

I

,;

I

Fraction

No.

Fig. 2. Polysomal patterns and radioactivity profiles in ethionine-treated and recovered livers.Polysomes were prepared f r o m ethionine-treated (5 h) and recovered livers as described under Materials and M e t h o d s with a use of Triton X-100. Sucrose density g~adient centrifugation was performed as given in the legend to Fig. 1. - - , A260nm; • . . . . . . •, radioactivity. Fractions A and B in (a) refer to the larger and smaller ribonucleoprotein particles to be analyzed in Fig. 5. (a) Ethionine treated. (b) Recovered.

310

[L 400

1.0

c

E

E

c o

u~

O (9

200

I

10

20

"°-o.°. ~

30 Fraction

10

20

30

No.

Fig. 3. T h e e f f e c t o f m i l d r i b o n u c l e a s e t r e a t m e n t on t h e s e d i m e n t a t i o n p a t t e r n s o f p o l y s o m e s a n d h e t e r o d i s p e r s e r a d i o a c t i v e m a t e r i a l s . T o e a c h 2 m l o f p o l y s o m a l s u s p e n s i o n s (2 m g r i b o s o m e s p e r m l o f t h e homogenizing medium), 0.001 pg of ribonuclease A (Sigma Type XI-A from bovine pancreas) was added and i n c u b a t e d at 3 7 ° C f o r 5 m i n . T h e m i x t u r e s w e r e t h e n c e n t r i f u g e d in t h e s a m e c o n d i t i o n s as d e s c r i b e d in the l e g e n d to Fig. 1. - - , A260nm; • ...... e, r a d i o a c t i v i t y , (a) E t h i o n i n e t r e a t e d . (b) R e c o v e r e d .

polysomes took place and the radioactivity profile essentially followed the absorbance profile of the remaining polysomes. On the other hand, when the same ribonuclease treatment was applied to the non-recovered ribosomes, the heterodisperse radioactive materials had largely disappeared (Figs 3a and 3b). These observations suggest that the heterodisperse radioactive materials are far more vulnerable to ribonuclease attack than the polysome-associated m R N A and resemble the rapidly labeled cytoplasmic ribonucleoprotein particles described by some investigators [22,24]. In order to confirm this idea, the heterodisperse radioactive materials and the recovered polysomes were centrifuged to equilibrium in CsC1 gradient after fixation with formaldehyde. The CsC1 density gradient analysis of the ribosomes from the ethionine-treated liver revealed two major peaks of radioactivity (Fig. 4a). A heavier peak (p = 1.55) which is at the shoulder of the A260 nm ribosome peak represents the polysomal m R N A and a lighter peak (p = 1.42) seems to represent the mRNA-containing ribonucleoprotein particles described by others [23, 25]. Thus we may conclude that in the cytoplasm of the ethionine-treated liver, most of the m R N A exists in the form of ribonucleoprotein particles, not associated with the ribosomes. When the same CsC1 density gradient analysis was performed on the recovered ribosomes, the polysomal peak had increased at the cost of the decrease in the ribonucleoprotein fraction (Fig. 4b), indicating that the ribonucleoprotein particles served as the precursors to the polysome reformation. That the fraction at p = 1.55 in fact represents the polysomal m R N A was confirmed by examining its sensitivity to EDTA. EDTA is known to quantitatively dissociate polysomes into subunits and to release b o u n d m R N A [26]. As is shown in Fig. 4c, the EDTA treat-

311

1.0

1.6

-

1.5

1,6

1.6

1,5

1,5

5O

C

1.4

1.4

i

!

13J

I

1.3

::

i~

~,~

u~ .J C

i

,%

o 25

0.5 .

i i

0

v

i

,~i.i.i*~-B-~" I

10

20

3O

10

20

Fraction

30

10

20

30

No,

Fig. 4. CsCl d e n s i t y a n a l y s i s o f h e t e r o d i s p e r s e r a d i o a c t i v e m a t e r i a l s f r o m e t h i o n i n e - t r e a t e d a n d r e c o v e r e d livers. P o l y s o m a l s u s p e n s i o n a n d t h e E D T A - t r e a t e d ( 2 0 r a M ) p o l y s o m a l s u s p e n s i o n f r o m t h e r e c o v e r e d liver w e r e f i x e d w i t h f o r m a l d e h y d e (6%). A f t e r d i a l y s i s a g a i n s t 50 m M t r i e t h a n o l a m i n e • H C l ( p H 7 . 6 ) / 5 0 m M KCl/1 m M MgCI 2 ( o r 1 m M E D T A in t h e case o f E D T A - t r e a t e d p o l y s o m e s ) , t h e f i x e d p o l y s o m a l p r e p a r a t i o n s w e r e a n a l y z e d o n CsCl g r a d i e n t s as d e s c r i b e d u n d e r M a t e r i a l s a n d M e t h o d s . • -=, A260rim; • ...... ¢, r a d i o a c t i v i t y . (a) E t h i o n i n e t r e a t e d . ( b ) R e c o v e r e d (c) R e c o v e r e d E D T A t r e a t e d .

ment of the recovered ribosomes quantitatively converted the 1.55 radioactive materials to the 1.42 particles, whereas the original 1.42 particles were essentially unaffected. Although we have so far established that the m R N A in the ethioninetreated liver exists mostly in the form of the ribonucleoprotein particles, the sucrose gradient sedimentation pattern reveals highly heterodisperse distribution with respect to the size of these particles. This may result from the difference in the relative amounts of R N A and proteins in the ribonucleoprotein particles. In order to check this possibility, the ribonucleoprotein particles were divided into the larger fraction (Fraction A) and the smaller fraction (Fraction B) in the sucrose density sedimentation as indicated in Fig. 2a. The pooled fractions were respectively submitted to the CsC1 b u o y a n t density analysis. As is shown in Fig. 5, the distribution of the b u o y a n t densities was found to be essentially the same for both classes of ribonucleoprotein particles, suggesting that the RNA/protein ratio is n o t much different between the larger and the smaller particles. This may be explained if the larger particles contain larger molecule of mRNA. The size distribution of m R N A in particles of different sizes was therefore investigated.

Characterization o f R N A extracted from ribonucleoprotein particles o f ethionine-treated livers The ribonucleoprotein particles in the cytoplasm of ethionine-treated liver were sedimented in sucrose gradient centrifugation in the same condition as shown in Fig. lc. The fractions representing various sizes of ribonucleoprotein particles were pooled and the particles were adsorbed on Millipore filters from which R N A was extracted by the sodium dodecylsulfate-phenol method. The R N A was then subjected to a sucrose gradient centrifugation analysis. As shown in Fig. 6, different size classes of ribonucleoprotein particles from Tri-

b

1,0

p

~.6

~

5O

1.6

1.5 E c o c~ <

%4

1.4

1.3

1,3

.c E u~ c

'.,

0,5

•!

/

10

20

30 Fraction

10

20

30

No.

Fig. 5. CsCl d e n s i t y a n a l y s i s o f t h e l a r g e r a n d t h e s m a l l e r h e t e r o d i s p e r s e r a d i o a c t i v e m a t e r i a l s f r o m e t h i o n i n e - t r e a t e d livers. T h e l a r g e r a n d t h e s m a l l e r h e t e r o d i s p e r s e r a d i o a c t i v e m a t e r i a l s w e r e f r a c t i o n a t e d , as s h o w n in Fig. 2. T h e p o o l e d f r a c t i o n s w e r e f i x e d w i t h f o r m a l d e h y d e a n d a n a l y z e d on CsCl d e n s i t y g r a d i e n t s as d e s c r i b e d in t h e l e g e n d to Fig. 4. -" o, A 2 6 0 n m ; • . . . . . . 0, r a d i o a c t i v i t y . (a) T h e l a r g e r f r a c t i o n , c o r r e s p o n d i n g to F r a c t i o n A o f Fig. 2a. (b) T h e s m a l l e r f r a c t i o n , c o r r e s p o n d i n g to F r a c t i o n B o f Fig. 2b. C

28S

18S

4S

28S

18S

4S

28S 18S

4S

o 0 25

10

20

30

._c

i

3'0

10

20

i

30

e

d 50

10 210 Fraction No.

28S

18S

4S

285

18S

4S

v

E m £3 i

25

/

3JO Fraction

i

i

10

20

310

No.

Fig. 6. S e d i m e n t a t i o n p a t t e r n s o f R N A e x t r a c t e d f r o m d i f f e r e n t size classes o f h e t e r o d i s p e r s e r a d i o a c t i v e m a t e r i a l s in e t h i o n i n e - t r e a t e d livers. D i f f e r e n t size classes of heterodisia'erse r a d i o a c t i v e m a t e r i a l s o b t a i n e d by s u c r o s e g r a d i e n t c e n t r i f u g a t i o n o f T r i t o n X - 1 0 0 - t r e a t e d or d e o x y c h o l a t e - t r e a t e d p o s t - m i t o c h o n d r i a l s u p e r n a t a n t w e r e p o o l e d as i n d i c a t e d in Fig. l c . R N A w a s e x t r a c t e d f r o m v a r i o u s f r a c t i o n s as d e s c r i b e d under Materials and Methods and c e n t r i f u g e d on a linear 5--20% sucrose gradient containing 100 m M NaCl, 1 m M E D T A a n d 25 m M T r i s • H C I ( p H 7 . 6 ) in a H i t a c h i SW-40 r o t o r a t 39 0 0 0 r e v . / m i n f o r 4 . 5 h. A r r o w s i n d i c a t e a p p r o x i m a t e S v a l u e s d e t e r m i n e d b y c a r r i e r R N A . a, F r a c t i o n A (Fig. l c ) , T r i t o n X - 1 0 0 t r e a t e d ; b, F r a c t i o n B, T r i t o n X - 1 0 0 t r e a t e d ; e, F r a c t i o n C, T r i t o n X - 1 0 0 t r e a t e d ; d, F r a c t i o n B, d e o x y c h o l a t e t r e a t e d ; e, F r a c t i o n C, d e o x y c h o l a t e t r e a t e d .

313

ton X-100-treated cytoplasm yielded radioactive RNAs with similar distributions of S values, showing a broad peak at around 10 S. The deoxycholatetreated ribonucleoprotein particles exhibited a similar pattern. These profiles of radioactive RNAs are very similar to those of cytoplasmic polysomal mRNA reported earlier [27,28], indicating that the RNA associated with the ribonucleoprotein particles is originated from the polysomal mRNA. The fact that the size of RNA derived from ribonucleoprotein particles is independent of the size of particles in the Triton X-:t00-treated cytoplasm strongly suggests that the larger ribonucleoprotein particles found in the cytoplasm of the ethioninetreated liver are the aggregates of smaller particles. The disappearences of the larger ribonucleoprotein particles in the deoxycholate-treated cytoplasm must be due to disaggregation of those larger particles into smaller particles by deoxycholate. State of ribosomes in ethionine-treated livers The experiments so far described have established that the mRNA portion is dissociated from the polysomes to form ribonucleoprotein particles by the ethionine treatment and the ribosomes are found in the monomer region in the sucrose density gradient centrifugation. The functional state of these ethioninetreated ribosomes, however, has remained unknown. It has been shown by Falvey and Staehelin [29] that rat liver ribosomes obtained by preincubation of polysomes in a cell-free protein synthesizing system are dissociated into subunits at a KC1 concentration of 0.5 M in the presence of 1 mM Mg2÷. Untreated polysomes, or ribosomes obtained by ribonuclease treatment containing mRNA fragments and nascent polypeptide chains are not dissociated under similar conditions [30].

eos

4ds

2,0

1.0

Iy

: '..... ..'"

0

,

1

.o. 2tO

Fraction

",., 310

No.

Fig. 7. D i s s o c i a t i o n o f e t h i o n i n e - t r e a t e d r i b o s o m e s in high i o n i c s t r e n g t h . C o n t r o l a n d e t h i o n i n e - t r e a t e d r i b o s o m e ( 0 . 2 rag) w e r e s u s p e n d e d in 0 . 2 m l o f h i g h s a l t b u f f e r ( 5 0 0 m M KC1, 1 m M M g C I 2 a n d 5 0 m M T r i s - H C I , p H 7 . 6 ) and c e n t r i f u g e d o n l i n e a r 5 - - 2 0 % s u c r o s e g r a d i e n t s s u p p l e m e n t e d w i t h t h e s a m e ingredients. C e n t r i f u g a t i o n w a s p e r f o r m e d i n a H i t a c h i S W - 2 5 . 3 r o t o r a t 2 5 0 0 0 r e v . / m i n f o r 5 h a t 2 0 ° C. , ethionine-treated

ribosomes; ......

control ribosomes.

314

Fig. 7 shows that exposure of the ethionine-treated ribosomes obtained after 5 h of ethionine injection to 0.5 M KC1 lead to almost complete dissociation into subunits while the same treatment of the control ribosomes yielded very small a m o u n t of subunits. Thus it appears that the ribosomal monomers in the ethionine-treated livers are active 'run-off' ribosomes containing no peptidyl-tRNA. That the ethionine-treated ribosomes are active in a cell-free protein synthesizing system was reported by Staehelin [31]. Discussion The present study shows that the administration of ethionine to rats causes the dissociation of hepatic polysomes into heterodisperse mRNA • protein complex and monomeric ribosomes. When the animals are recovered from the ethionine t r e a t m e n t by subsequent administration of adenine and methionine, the heterodisperse ribonucleoprotein particles and monomeric ribosomes appear to be utilized for the reformation of polysomes in the liver. Similar phenomena of the polysome breakdown and recovery have been described in the nutritional shiftdown and shiftup in the culture of mouse sarcoma 180 ascites cells [23] and also in the temperature shock and recovery in the cultured L cells [24]. In all these cases, m R N A appears to exist as heterodisperse messenger ribonucleoprotein particles after the polysome breakdown. The existence of free cytoplasmic m R N A • protein complexes has also been reported in HeLa cells [32] and in chicken embryos [33]. The c o m m o n feature of these ribonucleoprotein particles is that they represent a temporary non-translatable form of mRNA. When the protein synthesis is resumed in the cells, those ribonucleoprotein particles will combine with the existing ribosomes and form functional polysomes. The association of m R N A with proteins in the form of ribonucleoprotein particles may protect m R N A from the nucleolytic attack. The present demonstration that mRNA exists as ribonucleoprotein particles in the ethionine-treated liver substantiates our earlier proposal [13] that the administration of ethionine causes a prolongation of the half-life of liver mRNA. The ribonucleoprotein particles found in the Triton X-100-treated cytoplasm of the ethionine-treated liver exhibit heterodisperse distribution of very high S values, well extending over the region of heavier polysomes. Since different size classes of these particles yield RNA with the same distribution of S values and show the same distribution of the b u o y a n t densities in CsC1 gradient centrifugation, we conclude that heavier ribonucleoprotein particles are the aggregates of smaller particles. These large molecular particles are apparently disaggregated into smaller particles by deoxycholate treatment. Whether these large molecular ribonucleoprotein particles exist as such in vivo or represent artifacts arising from the isolation procedures remains to be established. A similar p h e n o m e n o n of reduction in the size of cytoplasmic ribonucleoprotein particles by deoxycholate t r e a t m e n t has been described [34]. Although ethionine exerts various metabolic effects on rat liver, the drastic reduction in the intracellular ATP concentration seems to be the primary event [4--6]. It remains to be explained, however, why the reduction in the intracellular ATP c o n t e n t leads to a specific breakdown of polysomes. More recent studies [ 21,35] indicate that ethionine inhibits hepatic protein synthesis

315

by reducing the rate of initiation. In view of the rapidity of polysome breakdown by ethionine and its reformation by adenine, one may assume that the low ATP concentration affects some labile factor concerned with the initiation of protein synthesis. Studies are n o w in progress to elucidate the nature of this labile factor.

Acknowledgement This work was supported by a grant from Ministry of Education, Japan.

References 1 2 3 4 5 6 7 8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

Villa-Trevino, S., S h u n , K.H. a n d F a r b e r , E. ( 1 9 6 6 ) J. Biol. C h e m . 2 4 1 , 4 6 7 0 - - 4 6 7 4 F a t h e r , E. a n d C o r b a n , M.S. ( 1 9 5 8 ) J. Biol. C h e m . 2 3 3 , 6 2 5 - - 6 3 0 F a t h e r , E., S i m p s o n , M.V. a n d Tarver, H. ( 1 9 5 0 ) J. Biol. C h e m . 1 8 2 , 9 1 - - 9 9 F a r b e r , E., Shull, K.H., Villa-Trevino, S., L o m b a r d i , B. a n d T h o m a s , M. ( 1 9 6 4 ) N a t u r e 2 0 3 , 3 4 - - 4 0 Shull, K . H . ( 1 9 6 2 ) J. Biol. C h e m . 2 3 7 , 1 7 3 4 - - 1 7 3 5 ViUa-Trevino, S., Shull, K.H. a n d F a r b e r , E. ( 1 9 6 3 ) J. Biol. C h e m . 2 3 8 , 1 7 5 7 - - 1 7 6 3 Villa-Trevino, S. a n d F a t h e r , E. ( 1 9 6 2 ) Bioehim. B i o p h y s . A e t a 6 1 , 6 4 9 - - 6 5 1 Villa-Trevino, S., F a r b e r , E., Staehelin, T., W e t t s t e i n , F.O. a n d Noll, H. ( 1 9 6 4 ) J. Biol. C h e m . 2 3 9 , 3826--3838 BagJio,C. and Farber, E. (1965) J. Mol. Biol. 12,466--467 ShuU, K.H. and Villa-Trevino,S. (1964) Biochem. Biophys. l~es.Commun. 16,101--105 Farber, E., Shull, K.H., MeConomy, J.M. and Castillo, A.E. (1965) Biochem. Pharmacol. 14,761--767 Stewart, G.A. and Farber, E. (1967) Science 157, 67--69 Endo, Y., Seno, H., Tominaga, H. and Natori, Y. (1973) Biochim. Biophys. Aeta 299, 114--120 Wettstein, F.O., Staehelln, T. and Noll, H. (1963) Nature 197,430--435 Brunk, C.F. and Leick, V. (1969) Biochim. Biophys. Acta 170,136--144 Iffet, J.B., Voet, O.H. and Vinogard, J. (1961) J. Phys. Chem. 65, 1138--1145 Infante, A.A. and Nemer, M. (1968) J. Mol. Biol. 32, 543--565 Lee, S.Y., Mendecki,J. and Brawerman, G. (1971) Proe. Natl. Acad. Sei. U.S. 68, 1331--1335 Brawerman~G., Mendeeki,J. and Lee, S.Y. (1972) Biochemistry i i , 637--641 Palaeios, R., Sullivan, D., Summers, N.M., Kiely, M.L. and Sehimke, R.T. (1973) J. Biol. Chem. 248, 540--548 Farber, E., Kisilevsky, R., ShuU, K.H. and Shinozuka, H. (1972) Advance in Enzyme Regulation (Weber, G., ed.), Vol. 10, pp. 383--394, Pergamon Press, New York Ishikawa, K., Kuroda, C. and Ogata, K. (1969) Biochim. Biophys. Acta 179,316--331 Lee, S.Y., Krsmanovic,V, and Brawerman, G. (1971) Biochemistry 10,895--900 Schochetman, G. and Perry, R.P. (1972) J. Mol. Biol. 63,577--590 Perry, R.P. and Kelly, D.E. (1968) J. Mol. Biol. 35, 37--59 Gros, F., Hiatt, H.H., Gilbert, W., Kurland, C.G., Risebrough, R.W. and Watson, J.D. (1961) Nature 190, 581--585 Tominaga, H., Aki, J. and Natori, Y. (1971) Biochim. Biophys. Aeta 228,183--192 Sugano, H., Suda, S., Kawada, T. and Sugano, I. (1971) Bioehim. Biophys. Aeta 238,139--149 Falvey,A.K. and Staehelin, T. (1970) J. Mol. Biol. 53, 1--19 Lawford, G.R. (1969) Bioehem. Biophys. Res, Commun. 37, 143--150 Staehelin, M. (1969) Biochim. Biophys, Acta 174,713--721 Spohr, G., Granboulan, N., Morel, C. and Scherrer, K. (1970) Eur. J. Bioehem. 17,296--318 Knbchel, W. and Tiederman, H. (1972) Biochim. Biophys. Aeta 269,104--117 Cartouzou, G.0 Poir~e, J.C. and Lissitzky, S. (1969) Eur. J. Biochem. 8,357--369 Kisilevsky,R. (1972) Biochim. Biophys. Acta 272,463--472