Different susceptibility of protein synthesis to inhibitors of elongation in cell-free systems from plasma cell tumours and reticulocytes

460

Biochimica et Biophysica Acta, 447 (1976) 460--473 © Elsevier/North-Holland Biomedical Press

BBA 98732

D I F F E R E N T SUSCEPTIBILITY OF PROTEIN SYNTHESIS TO INHIBITORS OF ELONGATION IN CELL-FREE SYSTEMS FROM PLASMA CELL TUMOURS AND RETICULOCYTES

B. EMMERICH, H. HOFFMAN, V. ERBEN and J. RASTETTER Hiimatologische A bt. der I. Med. Klinik der Technischen Universita't Miinchen, 800 Miinchen 80, Ismaninger Str. 22 (G.F.R.)

(Received December 5th, 1975) (Revised manuscript received June 15th, 1976)

Summary Plasma cells and reticulocytes are mammalian cell systems which have specialized in the synthesis of a single protein during their differentiation from one c o m m o n stem cell. T o study whether there is a difference in cell susceptibility at the level of elongation, dose vs. inhibition curves of sparsomycin, cycloheximide and emetine in cell-free systems with S-30 fractions from plasma cell tumours (MOPC 63, MOPC 41, RPC 20, MOPC 104 E), reticulocytes and liver were compared. The experiments revealed: (1) all the selected systems are equally sensitive to sparsomycin; (2) the susceptibility of the reticulocyte systems to cycloheximide and emetine is higher than that of the plasma cell tumours. In the dose range of 1 • 10 - 7 - 5 • 10 -s M cycloheximide and 1 • 10 - 6 - 1 • 10 -4 M emetine the reticulocyte system is preferentially inhibited; (3) the sensitivities of all plasma cell tumours are equal; (4) the liver system is more sensitive to emetine than to cycloheximide; (5) the site of the different susceptibility to these antibiotics could be located on the ribosomes; (6) however, when the extracts of the plasma cell tumours were prepared in the presence of hemin, their susceptibility rises and is like that of reticulocytes. These results show that hemin promotes in a cell-specific manner the sensitivity to some inhibitors of protein synthesis.

Introduction Inhibitors of protein synthesis are widely used as tools to elicit structural differences in several sites of the protein-synthesizing machinery between prokaryotic and eukaryotic cells [1,2]. In addition, differences in eukaryotic ribosomes have been detected by the selective action of tenuazonic acid. Carrasco and Vazquez [3] have shown that this antibiotic inhibits the peptidyltrans-

461 ferase centre of mammalian ribosomes but has no effect on yeast ribosomes. The purpose of this study was to investigate the susceptibility of different mammalian cells to inhibitors of protein synthesis to see whether there are also differences in mammalian ribosomes. The mammalian cells selected were reticulocytes, plasma cell tumours and liver. The plasma cells and the reticulocytes are the last stages of differentiation from one common stem cell [4--6]. They are characterized with respect to protein synthesis by their specialization for the synthesis of one distinct protein and by a differently developed endoplasmic reticulum, typical of secretory cells and non-secretory cells, respectively. The hepatic cells are specialized for the synthesis of different proteins and their cytoplasm has a highly organized endoplasmic reticulum similar to that found in plasma cell tumours [7]. To study whether there is a difference in cell susceptibility at the level of elongation, dose vs. inhibition curves of inhibitors in cell-free systems from these cells were compared. Cycloheximide, emetine and sparsomycin were used as inhibitors of elongation [8--10]. The experiments showed that protein synthesis in cell-free systems from reticulocytes is 1000 times more sensitive to cycloheximide and 100 times more sensitive to emetine than that in plasma cell tumour, cell-free systems. The low sensitivity of the plasma cell tumour system could be overcome when the cell extracts were prepared in the presence of hemin. Independent of the presence or absence of hemin all the selected systems are equally sensitive to sparsomycin. Materials and Methods 1. Chemicals. [U-14C]leucine (348 Ci/mmol)and [U-14C]phenylalanine (498 Ci/mmol) were obtained from the Radiochemical Centre, Amersham, U.K. Poly(U), ATP, GTP, creatine phosphate, creatine phosphokinase and cycloheximide were purchased from Boehringer Mannheim. Emetine was obtained from Sigma Chemical Co. and sparsomycin produced by Upjohn was a generous gift of Dr. W. Siegert. 2. Plasma cell tumours, reticulocytes and liver. The mouse plasma cell tumours strains MOPC 41, MOPC 63 {both k-type, L-chain producers), RPC 20 (),-type, L-chain producer) and MOPC 104 E (IgM with k-type, L-chain producer) were kindly provided by Dr. M. Potter (National Institutes of Health, U.S.A.) and Dr. B. Mach (D~partement de Biologie Mol~culaire, Universit~ de Gen~ve). Tumour lines were maintained in female inbred BALB/c mice by serial transplantation performed by subcutaneous injection of turnout fragments prepared in Medium 199 (Difco). Turnouts were removed immediately after killing the mice and either used immediately for preparation of the cell-free extracts or frozen in liquid nitrogen and stored at --20°C. Reticulocytes were obtained from phenylhydrazine-injected rabbits (institute strain) [11]. Liver were removed from male wistar rats (approx. 200 g), which were sacrificed after 16 h starvation. 3. Preparation o f the cell-free extracts. Rabbit reticulocyte postmitochondrial supernatant (S-30) was routinely prepared without chromatography on Sephadex G-25 as described by Schreier and Staehelin [11 ]. S-30 from the plasma cell tumours and livers was prepared according to Mathews and Korner

462 [12] with the following modifications: tumours and livers were weighed and scissor minced in 2.5 vol. of cold 10% sucrose in medium M containing 5 mM MgC12/80 mM KC1/20 mM Tris • HC1 pH 7.5/6 mM 2-mercaptoethanol. The mince was homogenized with 5 strokes of a motor-driven Potter homogenizer with a loose-fitting teflon pestle. The homogenate was centrifuged at 15 000 X g for 15 min and the resulting supernatant recentrifuged at 15 000 )< g for 15 min. The postmitochondiral supernatant was then passed through a Sephadex G-25 (coarse) column, equilibrated with medium M. The void fraction was collected in small aliquots and frozen in liquid nitrogen. In the experiments, in which the effect of hemin was tested, 50 pM hemin was added to the homogenization buffer. A stock solution of 1 mM hemin was prepared by dissolving hemin in 1/100 vol. of 1 M KOH, followed by 0.6 vol. of 20 mM HEPES buffer (pH 7.4) and 0.39 vol. of water. The ribosome concentration of each S-30 preparation was determined according to the modified m e t h o d of Mathews and Korner [12]: 5 ml of S-30 were made 1% in sodium deoxycholate and the ribosomes from two aliquots of 2.2 ml were pelleted by centrifugation at 15 000 X g for 3 h through a 5 ml layer of 1 M sucrose dissolved in medium M. The ribosomes of the resuspended pellet were measured assuming 1 A260nm = 80 pg ribosomes = 16 pmol [11,13]. The ribosome concentration of one S-30 preparation determined by this method was within + 5% of the mean value. The polysomes from rabbit reticulocytes, mouse plasma cell tumours and rat liver were prepared according to Schreier and Staehelin [ 11 ]. The yield of polysomes from reticulocytes was 1.5--2.5, from plasma cell tumours 1.0--1.5 and from liver 2.0 mg/g tissue, pH 5 enzymes were prepared from rat liver, RPC 20 plasma cell t u m o u r and reticulocytes according to Falvey and Staehelin [15]. All preparations were stored in liquid nitrogen. The protein concentration was determined by the m e t h o d of Lowry et al. [14]. 4. In vitro translation o f the endogenous messengers with S-30 extracts or p o l y s o m e s and p H 5 e n z y m e s from the different tissues. The standard reaction mixture of the S-30 system contained per ml: 500 pl S-30 extract (containing 2.5--18 A260nm units of ribosomes), 1 #mol ATP, 0.1 pmol GTP, 2 mg creatine phosphate, 0.2 mg creatine phosphokinase, 20 #mol Tris • HC1 pH 7.5, 3 pmol magnesium acetate, 80 pmol KC1, 6 pmol of 2-mercaptoethanol, 50 nmol of 20 L-amino acids except leucine, 200 pl ['4C]leucine (= 10 pCi = 28 nmol). In the experiments in which the effect of hemin was tested, the final hemin concentration was 50 pM. The standard reaction mixture of the polysomes and pH 5 enzymes system contained per ml: 200--400 pl polysomes (corresponding to 20--40 AE60nm units polysomes) and 200 pl pH 5 enzymes (approx. 5 mg protein). The final ionic conditions were 100 mM NH4CI, 3 mM magnesium acetate, 30 mM Tris • HCI pH 7.5 and 1 mM dithiothreitol. All the other components were the same as in the S-30 systems. Reaction mixtures, generally 0.15 ml, were incubated at 30 or 37°C. Aliquots of 20 pl were pipetted at zero time and at intervals up to 30 min and protein synthesis assayed with the Mans-Novelli filter-disc technique [ 16]. Radioactivity was counted in 5 ml PPO/POPOP/toluol scintillation mixtures in a scintillation counter (Packard) at 80% efficiency for all systems except the

463 S-30 system from reticulocytes. In this system the efficiency was reduced to 30% due to quenching by hemoglobin. Analysis of the in vitro-synthesized products was carried out with sodium dodecyl sulphate-acrylamide gel electrophoresis and autoradiography of the [3SS]methionine-labeled peptides according to Laemmli [17,18]. We thank Dr. W. Siegert for performing the gel electrophoresis. 5. Comparison of the susceptibility. The susceptibility of protein synthesis in the in vitro systems of the different cells was compared using dose vs. inhibition curves. In these curves the decrease of the incorporation compared with the control, expressed as percentage during the time of linear incorporation, and the molar concentration of inhibitor were used as units of inhibition and doses respectively. Results

1. Endogenous translation in the in vitro systems The activities of the different in vitro incorporation systems are summarized in Table I. The systems from reticulocytes were more active than the other systems. The cell-free systems synthesized a limited number of well-distinguishable endogenous proteins (Fig. 1). On the autoradiographs from the reticulocyte S-30 cell-free products the main band of radioactivity was located in the molecular weight region of the a- and ~-globin chain (mol. wt. 15 × 103). MOPC 63 S-30 produced distinguishable, but low-labeled, bands in the region where immunogiobulin light chains (mol. wt. 20 × 103) would be expected. 2. Influence of the ribosome concentration of the cell-free systems on the inhibition effect Determination of the ribosome concentration in the S-30 extract from the different cells revealed that the ribosomes in the S-30 fraction of the reticulocytes were only 1/5--1/6 of the concentration which was found in the other S-30 extracts. This is due to a higher dilution by lysis procedures during the reticulocyte S-30 preparation. Since the drugs are considered to act on the ribosome, their effect may be influenced by these different ribosome concentrations in the S-30 cell-free systems. To exclude different sensitivities caused by different ribosome concentrations of the S-30 extracts, the influence of the ribosome concentration in the

TABLE

I

ACTIVITIES OF THE DIFFERENT

IN V I T R O

INCORPORATION

SYSTEMS

Activity is expressed as p m o l leucine incorporated per p m o l ribosome in 30 m i n at 37°C. For further details see Materials a n d M e t h o d s .

S-30

Reticuloeytes

MOPC 63

MOPC 41

RPC 20

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1.0

1.0

2.3

2.1

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0.5

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1.5

2.5

464

Fig. 1. A u t o r a d i o g r a p h s o f the e n d o g e n o u s cell-free p r o d u c t s in the S-30 s y s t e m s f r o m m o u s e p l a s m a cell t u m o u r MOPC 63 (Nos. 1 1 5 ) , r a b b i t r e t i c u l o c y t e s (Nos. 6 - - 8 ) , a n d r a t liver (Nos. 9 - - 1 1 ) . S t a n d a r d protein s y n t h e s i s r e a c t i o n s ( 5 0 pl) c o n t a i n e d 50 pCi 35S m e t h i o n i n e ( 2 5 0 C i / m m o l ) = 0 . 2 5 n m o l plus nine: t e e n u n l a b e l e d a m i n o acids (2.5 n m o l each). I n c u b a t i o n was 6 0 rain at 3 7 ° C . T h e p r o t e i n s o f 30-pl samples w e r e a p p l i e d t o t h e gel Nos. 1--5, 6 a n d 9. T h e s a m p l e s f o r gel Nos. 7,8 a n d 1 0 , 1 1 w e r e d i l u t e d I : 2 a n d 1 : 4. T h e m o l e c u l a r w e i g h t s t a n d a r d s w e r e b o v i n s e r u m a l b u m i n (tool. wt. 6 0 X 1 0 3 ) , r a b b i t aldolase ( 4 0 X 1 0 3 ) , c h y m o t r y p s i n o g e n A ( 2 5 X 1 0 3 ) , h o r s e m y o g l o b i n ( 1 7 . 8 X 1 0 3 ) . F o r f u r t h e r details see Materials a n d M e t h o d s a n d ref. 18.

reaction m i x t u r e on the inhibitory e f f e c t of the drugs was tested. Fig. 2 shows the results for cycloheximide. Increasing the ret i cul ocyt e or plasma cell t u m o u r ribosome c o n c e n t r a t i o n did n o t change the inhibition by a given inhibitor concentration. Identical results were obtained with emetine, so that the inhibition o f the p r o t e i n synthesis by these drugs is n o t affected by variation of the ribosome c o n c e n t r a t i o n in the range f o u n d in the S-30 extracts. Hence, the ribosome c o n c e n t r a t i o n in the reaction m i xt ur e could be neglected when the sensitivities o f the several cell-free systems were com pared and the molar drug concentration could be used as a dose unit in the dose vs. inhibition curves.

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ribosomes ( A26010.1ml ) Fig. 2. I n h i b i t i o n o f p r o t e i n s y n t h e s i s b y c o n s t a n t c o n c e n t r a t i o n o f e y c l o h e x i m i d e a n d v a r i a b l e a m o u n t s o f p o l y s o m e s in t h e r e a c t i o n m i x t u r e . T h e r e a c t i o n m i x t u r e c o n t a i n e d p e r 0 , 1 ml: p o l y s o m e s o f r e t i c u l o c y t e s ( o - - - - - - - o ) a n d p o l y s o m e s o f R P C 2 0 (o o) as i n d i c a t e d ; p H 5 f r a c t i o n f r o m liver I 0 #l. All o t h e r c o m p o n e n t s w e r 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 . F o r e a c h P O l y s o m e c o n c e n t r a t i o n a complete kinetic experiment of the control and inhibitor-containing assay was performed. The cyclohexim i d e c o n c e n t r a t i o n w a s in all a s s a y s w i t h r e t i c u l o c y t e p o l y s o m e s 1 • 1 0 -5 M a n d w i t h R P C 2 0 p o l y s o m e s 1 • 1 0 -3 M, T h e i n h i b i t i o n is e x p r e s s e d as p e r c e n t a g e o f t h e c o n t r o l a f t e r a 1 0 r a i n i n c u b a t i o n .

3. Susceptibility of the endogenous incorporation to cycloheximide and emetine The time course of incorporation in the different S-30 systems, both in the presence and absence of different concentrations of cycloheximide, is shown in Fig. 3. In the control, protein synthesis was linear over a period of about 10

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min. At all concentrations the drug inhibited the incorporation immediately after the start of the reaction. This is expected by inhibition o f chain elongation [ 1 0 ] . The concentrations o f cycloheximide required to inhibit protein synthesis by 50% were 5 • 10 -6 M in the reticulocyte system and 1 • 10 -2 M in the MOPC 63 tumour system. The liver system showed only a 35% inhibition at 1 • 10 -2 M. The results from such kinetic experiments with cycloheximide and emetine are summarized in the dose vs. inhibition curves (Figs. 4 and 5). By these curves

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467 the sensitivity of protein synthesis in the different S-30 systems to both inhibitions could be compared. They demonstrate: (a) the susceptibility of the reticulocyte S-30 system to cycloheximide and emetine is much higher than that of the plasma cell tumours. Protein synthesis is inhibited 50% by 2 • 10 -6 M cycloheximide and 1 • 10 -s M emetine in the reticulocyte system, and at 5 • 10 -3 M cycloheximide and 2 • 10 -3 M emetine in the plasma cell t u m o u r system; (b) in the dose range of 1 • 10 -7 --5 • 10 -s M cycloheximide and 1 • 10 - 6 1 • 10 -4 M emetine the reticulocyte system is preferentially inhibited; (c) the dose vs. inhibition curves of all plasma cell tumours are very similar; (d) the liver system is the most insensitive system to cycloheximide. The susceptibility of the liver S-30 to emetine is much higher than to cycloheximide, this dose vs. inhibition curve is identical with that of the reticulocyte S-30 system.

4. Localisation of the fraction responsible for the different susceptibility of the S-30 extracts The differential sensitivity of the reticulocytes and plasma cell t u m o u r S-30 systems to emetine and cycloheximide prompted the question as to whether factors of the supernatant also share in the different susceptibility or whether the polysome fractions alone are involved. To answer this question experiments were performed in which polysomes and pH 5 fractions of the sensitive and insensitive S-30 system were interchanged (Fig. 6). The results obtained in the homologous S-30 systems from reticulocytes and RPC 20 plasma cell tumour are demonstrated in Fig. 6A. Addition of the pH 5 fraction from the sensitive liver system to polysomes from reticulocytes and RPC 20 plasma cell tumour makes the RPC 20 polysomes 10 times more sensitive than the S-30 system of RPC 20, b u t they are still 100 times less sensitive as the reticulocyte polysomes, so that the difference between both systems is n o t markedly abolished (Fig. 6B). Identical results were obtained using pH 5 fraction from reticulocytes. Finally, when the reticulocyte polysomes were combined with the supernatant of low-sensitive RPC 20 plasma cell tumour, neither the activity nor the susceptibility of the reticulocyte polysomes is altered (Fig. 6C). In analogous cross experiments, similar results were obtained for cycloheximide. pH 5 fraction from liver, which exhibited low sensitivity to cycloheximide, was added to polysomes from reticulocytes and plasma cell tumours. The doses, which caused an inhibition of 50--60% in this system, were for reticulocytes polysomes 1 • 10 -s M and for plasma cell t u m o u r polysomes 1 • 10 -3 M (see also Fig. 2). This difference corresponds to that found between the S-30 systems. These cross experiments demonstrate that only the polysome fraction is responsible for the different susceptibility to emetine and cycloheximide. To exclude the influence of different messenger R N A populations on susceptibility the inhibition of the poly(U)-directed phenylalanine incorporation by cycloheximide in the different S-30 systems was compared (Table III). The results obtained showed the same difference between the susceptibility of reticulocytes and plasma cell tumours and liver as seen in the cell-free systems programmed by endogenous messenger RNA. For practical reason S-30 and polysome fractions were prepared from different animal species. The reticulocytes were taken from rabbits, the liver from

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emetine -10g (M) Fig. 6. D o s e vs. i n h i b i t i o n c u r v e s o f e m e t i n e in t h e h o m o l o g o u s S - 3 0 a n d t h e m i x e d s y s t e m w i t h polys o m e s a n d p H 5 f r a c t i o n s ( c r o s s - o v e r e x p e r i m e n t s ) w i t h o u t a d d i t i o n o f h e r o i n . F o r c o n d i t i o n s o f cell-free p r o t e i n s y n t h e s i s w i t h S - 3 0 e x t r a c t s a n d p o l y s o m e s a n d p H 5 f r a c t i o n see M a t e r i a l s a n d M e t h o d s . ( A ) D o s e vs. i n h i b i t i o n c u r v e s o f t h e S - 3 0 s y s t e m s : o - o, r e t i c u l o c y t e s ; • • , liver, D - - D , RPC 2 0 p l a s m a cell t u r n o u t . (B) D o s e vs. i n h i b i t i o n c u r v e s o f t h e s y s t e m s w i t h p o l y s o m e s f r o m r e t i c u l o c y t e s (o o) a n d R P C 2 0 p l a s m a cell t u r n o u t (u D) m i x e d w i t h p H 5 f r a c t i o n f r o m l i v e r (liver s h o w e d h i g h s u s c e p t i b i l i t y in t h e S - 3 0 s y s t e m ) . ( C ) D o s e vs. i n h i b i t i o n c u r v e s o f p o l y s o m e s f r o m r e t i e u l o c y t e s (o----o) m i x e d w i t h p H 5 f r o m R P C 2 0 p l a s m a cell t u r n o u t ( R P C 20 s h o w e d l o w s u s c e p t i b i l i t y in t h e S-30 s y s t e m ) .

rats and the plasma cell t um our s from mice. Therefore, a species-specific effect f • c y clo h ex imid e and emetine m ay also a c c o u n t for the difference in sensitivity f o u n d between these cells. T o investigate w h e t h e r such an e f f e c t is involved, the inhibition o f the S-30 system from rabbit, rat and mouse livers was compared. The dose vs. inhibition curves o f all liver systems were identical.

5. Effect o f hemin on the susceptibility to cycloheximide and emetine Recently, Weber et al. [ 19] have r e p o r t e d t h a t HeLa cell extracts prepared in the presence o f hemin are m or e active in initiation of prot ei n synthesis than extracts prepared w i t h o u t hemin. In this system a remarkable shift t o a higher sensitivity to aurintricarboxylic acid after addition o f hemin was observed.

469 T A B L E II E F F E C T O F C Y C L O H E X I M I D E ON P O L Y P H E N Y L A L A N I N E FROM DIFFERENT MAMMALIAN SYSTEMS

SYNTHESIS

BY S-30 E X T R A C T S

R e a c t i o n m i x t u r e o f t h e p o l y ( U ) - d i r e e t e d p o l y p h e n y l a l a n i n e s y s t e m w i t h S-30 f r o m t h e d i f f e r e n t tissues c o n t a i n e d in 1.0 ml: 2 0 ~ m o l Tris • HCI ( p H 7 . 5 ) , 8 ~tmol MgCl 2 in t h e p l a s m a cell t u r n o u t a n d liver syst e m and 3.5 ~ m o l MgC12 in the r e t i c u l o c y t e s y s t e m , 1 0 0 ~umol KCi, 6 ~ m o l 2 - m e r e a p t o e t h a n o l , 1 Drool A T P , 0.1 ~umol G T P , 2 m g e r e a t i n e p h o s p h a t e , 0 . 2 m s c r e a t i n e p h o s p h o k i n a s e , 5 0 n m o l o f e a c h o f the 20 L - a m i n o acids e x c e p t p h e n y l a l a n i n e , 2 0 0 pl [ 1 4 C ] p h e n y l a l a n i n e ( 4 9 5 C i / m o l ) = 2 0 n m o l = 10/~Ci, 1 mE p o l y ( U ) , 4 0 0 ~l S-30, I n c u b a t i o n : 3 7 ° C for 3 0 rain. T h e i n c o r p o r a t i o n w i t h o u t p o l y ( U ) was in liver a n d p l a s m a cell t u m o u r s a b S u t 10% a n d in r e t i c u l o c y t e s u n d e r 5%, S-30

Cycloheximide (M)

[ 14 C ] P o l y p h e n y l a l a n i n e incorporation directed by poly(U) (cpm)

% of C o n t r o l

Rabbit reticulocytes

None

2150 842

i00 37

None

2420

100

2,5 • 10 -3 None 2,5 • 10 -3 None 2.5 • 10 -3

820 2100 720 1140 550

34 100 34 100 48

1 MOPC

63

R P C 20

R a t liver

• 10 -5

These findings promote us to carry out the inhibition experiments also in the presence of hemin. Addition of hemin to the cell-free system with S-30 from reticulocytes did not alter the effect of cycloheximide and emetine. This is consistent with results obtained also in while reticulocytes [20]. However, when plasma cell turnout extracts were prepared and incubated in the presence of heroin their sensitivity to both inhibitors rose to that of the reticulocyte system (see Figs. 5 and 6), whereas the activities of the controls were not affected.

100 O

r~

I

o

g 75

~d 50

~

nn/

o

• o •

ret~cu[ocytes liver RPC 20 ;~ Ichain • MOPC 104E /~ MOPC41 K Ichain

25

6

5 sparsomycin

- log

4 (M)

Fig. 7. Dose vs, i n h i b i t i o n c u r v e s o f s p a z s o m y c i n in t h e S-30 s y s t e m s f r o m r e t i c u l o c y t e s , P l a s m a t u m o u r s ( R P C 20, MOPC 1 0 4 E, MOPC 4 1 ) a n d liver. F o r f u r t h e r details see l e g e n d o f Fig. 3,

470

6. Susceptibility of the endogenous incorporation to sparsomycin Whereas emetine is considered to be an inhibitor of translocation [25,37] and cycloheximide as an inhibitor of translocation as well as transpeptidation [37,38], sparsomycin is regarded as a specific inhibitor of peptidyltransferase reactions. The sensitivity of the protein synthesis in S-30 from reticulocytes, the plasma tumours RPC 20, MOPC 104E and MOPC 41 and liver to sparsomycin is demonstrated in Fig. 7. The dose vs. inhibition curves are almost identical, indicating that all systems show the same susceptibility to sparsomycin. The sparsomycin concentration, at which the endogenous incorporation is inhibited by 50%, was 1 • 10 -6 M, corresponding to a molar ratio of sparsomycin to ribosome of a b o u t 1. Discussion

The purpose of this study was to compare the susceptibility of the protein synthesis to several inhibitors of elongation in different mammalian cells. For this the S-30 cell-free system was used. Protein synthesis in cell-free systems from reticulocytes, plasma cell tumours and liver are equally sensitive to sparsomycin. This indicates that the sites of the peptidyltransferase centre, which is blocked by this antibiotic, is similar in these mammalian cells. In contrast to this finding, protein synthesis in S-30 extracts from reticulocytes is 100--1000 times more sensitive to cycloheximide and emetine than protein synthesis in extracts from plasma cell tumours. Ribosome titration experiments revealed that the different ribosome concentrations in the extracts of the different cells could not account for these different sensitivities. Furthermore, an artefact due to freezing the tissues and G-25 chromatography of S-30, both routinely used for the plasma cell t u m o u r system, could be excluded. To localize the subcellular components which are responsible for the different sensitivities of the S-30 systems, cross experiments were done in which polysomes and supernatant fraction from the different cells were interchanged. The results indicate that the susceptibility is due to the polysomes fraction. Moreover, experiments in which the sensitivity of the protein synthesis with endogenous messenger RNA was compared with that of poly(U)-directed polyphenylalanine synthesis show that the different sensitivity is independent of the template and that only the ribosomes are responsible for the different sensitivity. This result is consistent with the observation that cycloheximide [21, 22] and emetine [23--25] interact with the 80-S ribosomes. Factors of the supernatant such as transferase II, which was suggested as one site of cycloheximide action [26--28] are n o t involved. An alteration of the susceptibility of plasma cell ribosomes by impurities with membranes is unlikely, since in the cross experiments polysomes were prepared in conditions under which only free polysomes were pelleted. In addition, the fact that the liver system has the same susceptibility to emetine as the reticulocyte system supports the assumption that different membrane contents of the cells or artifacts due to different preparation procedures of the polysomes are not the cause of the different susceptibility. To exclude a species-specific effect the sensitivities of the liver systems from

471 mouse, rat and rabbit were compared. The results show that there is only a specific sensitivity with respect to tissues and n o t to species. The finding that reticulocyte ribosomes are 100 times more susceptible to cycloheximide and emetine than plasma cell turnout ribosomes is remarkable, when one compares this with the observation that Escherichia coli ribosomes are only 30 times more sensitive to tetracycline and chlortetracycline then eukaryotic ribosomes [ 29]. The sensitivity of the reticulocyte in vitro system is not affected by the addition of exogenous heroin. This is consistent with observations in whole cells [20]. The original low sensitivity of the plasma cell tumours, however, can be overcome by addition of hemin to the preparation medium. This m a y be due to a lower endogenous hemin concentration in plasma cell tumours than in reticulocytes. However, in contrast to this assumption, the endogenous hemin concentration in reticulocytes is considered to be lower than in other tissues since hemin in these cells is incorporated into hemoglobin [30]. A preferential elimination of hemin by gel filtration on Sephadex G-25 could n o t be the cause of the different response of the plasma cell t u m o u r and reticulocyte systems to added hemin, because the sensitivity of S-30 extracts did n o t alter whether they were passed through Sephadex G-25 or not. Heroin is able to stimulate the initiation n o t only in reticulocytes b u t also in other mammalian cells by inhibiting the production of a translational repressor [19,30--33]. Cycloheximide and emetine are considered to inhibit, not only chain elongation b u t also chain initiation [10,28]. Hence, the higher sensitivity of t h e hemin-supplemented plasma cell t u m o u r system could be a result of increased initiation. Opposed to this supposition is that we did n o t find any indication for an increased initiation in the hemin-supplemented plasma cell tumour S-30 system. Neither the activity, the kinetics of incorporation, nor the sensitivity to aurintricarboxylic acid, an inhibitor which blocks initiation at low and elongation at high concentrations [10], were affected {results n o t shown). Moreover, an increased initiation can only account for the shift in sensitivity, when the drugs inhibit initiation at lower concentrations than elongation. Concerning this point the opinion~ a b o u t the action of cycloheximide and emetine are contradictory. Baglia et al. [28], like others [34,35], suggested from polysomes studies that initiation is inhibited b y lower concentration of cycloheximide rather than elongation. More recently however, Lodish et al. [10] concluded from their studies of the incorporation of formyl methionine into N-terminal positions of globin chains and that of methionine into internal positions, that cycloheximide and emetine in contrast to aurintricarboxyiic acid do not, at any concentration, function as selective inhibitors of initiation. These considerations support the suggestion that besides the effect of hemin on initiation an additional effect on elongation exists by which the increased susceptibility of the hemin-supplemented plasma cell t u m o u r system must be explained. The question whether this hemin effect is a direct effect on the ribosomes or like the effect on chain initiation transmitted b y factors which are able to inactivate the drugs was checked by the following experiments. Cycloheximide and emetine at concentrations which inhibit the reticulocyte system were incubated under conditions of protein synthesis with plasma cell t u m o u r extracts prepared with and w i t h o u t hemin. The incubation mixture was then centrifuged to

472 remove the plasma cell microsomes and after centrifugation reticulocyte polysomes were added to the supernatant and incubation was continued by adding fresh energy-regenerating system and labeled leucine. In these experiments plasma cell t u m o u r extracts with and without hemin did not abolish the extent of inhibition of both inhibitors on the reticulocyte polysome-dependent protein synthesis. These results favour the assumption that the susceptibility of plasma cell tumour ribosomes is directly affected by hemin. Whether this is caused by a physiological or nonphysiological interference with ribosomal components involved in the interaction with both drugs or by preserving the natural conditions of the ribosomes cannot be decided at present. The observations that protein synthesis in vivo in several cell systems is more sensitive to cycloheximide [36] and emetine [23] than protein synthesis in vitro support the last possibility. Furreading of the manuscript. The experr technical assitance of R. Baier is gratefully acknowledged.

Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft, grant Em 20/2. We thank Professor Dr. W. Kersten for helpful discussion and critical reading of the manuscript. The expert technical assistance of R. Baier is gratefully acknowledged.

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