Inhibitory effect of negamycin on polysomal ribosomes of Escherichia coli

406

Biochimica et Biophysica Acta, 447 (1976) 406--4~ 2 © Elsevier/North-Holland Biomedical Press

BBA 98738 INHIBITORY E F F E C T OF NEGAMYCIN ON POLYSOMAL RIBOSOMES OF E S C H E R I C H I A COLI

YOSHIMASA UEHARA, MAKOTO HORI and HAMAO UMEZAWA Institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo (Japan) (Received March 22nd, 1976) (Revised manuscript received July 20th, 1976)

Summary Protein synthesis with initiation-free polysomes from Escherichia coil was inhibited by negamycin at the termination step; release of peptides from ribosomes but n o t accumulation o f nascent peptides on ribosomes was inhibited. The result suggests t h a t negamycin, unlike streptomycin, binds to polysomal ribosomes as efficiently as to single ribosomes. The bactericidal effect of negamycin in the presence of chloramphenicol is discussed in this connection.

Introduction

Negamycin is a peptide like antibiotic [1] with strong miscoding activity [2,3]. We have reported t h a t negamycin inhibits preferentially the termination step of phage f2 RNA-directed protein synthesis in vitro [4] and that termination of protein synthesis must include a process which is susceptible to negamycin in a specific manner [5]. The present paper reports that protein synthesis with initiation-free polysomes from Escherichia coli [6] is inhibited by negamycin also at the termination step and that the binding between negamycin and polysomal ribosomes must be irreversible under physiological conditions because of the bactericidal action of negamycin in the presence of chloramphenicol. In this connection, one should remember that streptomycin, a typical miscoding antibiotic [7], has only a limited effect on polysomal ribosomes and lacks bactericidal action in the presence of chloramphenicol [8]. Materials and Methods Preparation o f initiation-free p o l y s o m e s Polysomes were prepared as described by Flessel et al. [9] with a minor modification. 40 ml culture of E. coli B was grown to a cell density of 2 • 10 s cells/ml and poured over an equal volume of crushed ice. The cells were har-

407 vested by centrifugation for 5 min at 10000 × g in a refrigerated centrifuge. The cell pellet was resuspended in 10 ml of 0.5 M sucrose/15 mM Tris • HC1, pH 7.8 (sucrose/buffer solution}. To the cell suspension, 1.0 ml of freshly dissolved lysozyme solution (2 mg/ml in sucrose/buffer solution) and 1.2 ml of 0.1 M EDTA (pH 8.0) were added. The mixture was left on ice for 3 min and 0.26 ml of 1 M MgSO4 was added. The spheroplast suspension was centrifuged for 5 min at 10000 × g. The supernatant was decanted out and the inside of the tube was wiped dry. The pellet was resuspended in 1.0 ml of lysing medium containing 10 mM Tris • HCI, pH 7.8, 10 mM magnesium acetate, 50 mM NH4C1, 0.5% Brij58, 0.5% sodium deoxycholate and 10 pg DNase I. Cytolysis was enhanced by gentle mixing with a glass rod. The lysate was t h e n centrifuged for 10 min at 12000 X g and the supernatant was carefully removed and layered over a discontinuous sucrose gradient consisting of 1 ml of 70% sucrose dissolved in buffer A (10 mM Tris • HC1, pH 7.8, 10 mM magnesium acetate, 50 mM NH4C1 and 6 mM 2-mercaptoethanol) and 3 ml of 30% sucrose in buffer A. The gradient was centrifuged for 17 h at 130000 × gay in a Hitachi RPS40T-2 rotor at 4 ° C. The pellet (purified polysomes) was suspended in 1 ml buffer A and stored at --90°C in small portions until use. Approx. 30 A260 was obtained from a 40 ml culture. These polysomes, free of ribosomal subunits, were stable during storage at --90 ° C.

Protein synthesis in vitro Protein synthesis with purified polysomes was carried out in a reaction mixture (0.1 ml) containing 50 mM Tris • HC1, pH 7.8, 60 mM NH4C1, 10 mM magnesium acetate, 3 mM ATP, 0.2 mM GTP, 2 mM phosphoenolpyruvate, 5 ~g of pyruvate kinase, 2 mM dithiothreitol, 0.03 mM ['4C]valine (50 Ci/mol, Daiichi Pure Chemicals), 19 other amino acids at 0.035 mM each, 30 ~g stripped tttNA, 100 ~g protein of 8100 extract and 0.6 A260 of purified polysomes. After incubation at 37°C for an indicated time, a p o r t i o n of reaction mixture was absorbed onto a paper disk and radioactivity in hot trichloroacetic acidinsoluble materials was determined as described previously [4].

Sucrose density gradient centrifugation analysis Protein synthesis was stopped by rapid chilling and addition of chloramphenicol at a concentration of 200 ug/ml [10]. A portion of each reaction mixture was gently layered over 4.9 ml of a linear sucrose density gradient (15--30% w/v in buffer A, less 2-mercaptoethanol) and centrifuged for 60 min at 120 000 × gay in a Hitachi RPS40T-2 rotor at 4°C. After centrifugation, the five-drop fractions were collected with continuous scanning for 254 nm absorption in an Ohtake gradient analyzer. Each fraction was m i x e d with 3 ml of cold 5% trichloroacetic acid and heated at 90°C for 20 min. Insoluble materials were filtered on a MiUipore HA filter disk (2.25 cm diameter, 0.45 pm pore size). After washing and drying, radioactivity trapped on a filter disk was measured in a liquid scintillation counter.

Viable cell counts E. coli B was grown in synthetic medium containing 0.2% glucose, 0.2% KH2PO4, 0.7% K2HPO4, 0.05% sodium citrate • 2H20, 0.1% (NH4)~SO4 and

408 0.01% MgSO4. The pH was adjusted to 7.4 with NaOH. The generation time was about 45 min in 10 ml cultures incubated at 37°C with gentle shaking. The antibiotics were added to the cultures during the logarithmic growth phase and the number of viable cells were determined at the time indicated by plating on nutrient agar after dilution with 0.85% NaC1. Results

Confirmation of initiation-free protein synthesis in a system with purified polysomes Kinetics of the protein synthesis with purified polysomes are shown in Fig. 1. Protein synthesis with this system was a linear function of time for the initial 3--4 min and leveled off by 20 min. Protein synthesis was dependent upon the concentrations of polysomes, supernatant factors and Mg 2÷. The optim u m range of magnesium concentration was broad, 8--13 mM, in contrast to 10 mM for the initiation-dependent protein synthesis directed by phage f 2 R N A (using NH4Cl-washed ribosomes, IFs, etc). The incorporation of amino acids into the acid insoluble fraction was inhibited completely by chloramphenicol, indicating that the incorporation was due to endogenous protein synthesis by polysomes. These characteristics resemble those of polysomes purified by Sepharose 4B column chromatography, established by Tai et al. [ 6 ] . The protein synthesis with purified polysomes was not inhibited by kasugamycin, a specific inhibitor of initiation [ 1 1 , 1 2 ] , as shown in Table I. In the table, the positive effect of kasugamycin on phage f2RNA-directed protein synthesis is shown for comparison. The result indicated that the former protein synthesizing system did not include significantly the initiation process (therefore, referred to as initiation-free protein synthesis).

20

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Fig. 1. Kinetics o f t h e p r o t e i n s y n t h e s i s w i t h p u r i f i e d p o l y s o m e s . R e a c t i o n m i x t u r e s ( 0 . 2 m l e a c h ) , w i t h (o) and w i t h o u t ( e ) p o l y s o m e s (1.2 A 2 6 0 ) , w e r e p r e p a r e d (see Materials and M e t h o d s ) and b o t h m i x t u r e s w e r e i n c u b a t e d at 3 7 ° C . A t t i m e s i n d i c a t e d , s a m p l e s ( 2 0 #1) w e r e w i t h d r a w n and r a d i o a c t i v i t y in h o t triehloroacetic acid-precipitable material was measured. Fig. 2. E f f e c t o~ Mg 2+ c o n c e n t r a t i o n o n t h e n e g a m y c i n a c t i o n . R e a c t i o n m i x t u r e s c o n t a i n i n g p u r i f i e d p n i y s o m e s , Mg 2+ at a desired c o n c e n t r a t i o n , e t c . w e r e i n c u b a t e d at 3 7 ° C for 1 0 rain e i t h e r w i t h 1 0 -4 M n e g a m y c i n ( e ) or w i t h o u t t h e a n t i b i o t i c ( o ) . T h e i n c o r p o r a t i o n in t h e c o n t r o l run ( w i t h o u t n e g a m y e i n ) at 10 m M Mg 2+ w a s t a k e n as 1 0 0 % a c t i v i t y . ( X ) s h o w s % i n h i b i t i o n .

409 TABLE I CONFIRMATION GAMYCIN

OF INITIATION-FREE

PROTEIN SYNTHESIS: LACK OF INHIBITION BY KASU ~

[ 1 4 C ] V a l i n e i n c o r p o r a t i o n b y i n i t i a t i o n free p o l y s o m e s and under t h e d i r e c t i o n o f f 2 R N A w a s a s s a y e d as d e s c r i b e d u n d e r Materials a n d M e t h o d s . T h e i n c o r p o r a t i o n in t h e c o n t r o l run ( w i t h o u t k a s u g a m y c i n ) was t a k e n as 1 0 0 % a c t i v i t y . T h e rate o f i n h i b i t i o n w a s c o n s t a n t t h r o u g h the i n c u b a t i o n t i m e in e a c h case. C o n c e n t r a t i o n of k a s u g a m y e i n (M)

2 - 10 -5 10 -4 10 -3

Percent inhibition Protein synthesis with purified polysomes

Phage f 2 R N A - d i r e c t e d protein synthesis

0 0 1

40 85 --

Inhibition by negamycin of the initiation-free protein synthesis As shown in Fig. 2, the inhibition by negamycin of the protein synthesis using purified polysomes was constant at various Mg 2÷ concentrations. The inhibition was partial and not increased b y increasing the concentration of negamycin from 10 -4 to 10 -3 M (data not shown). This was consist with the result obtained in a protein synthesizing system including the initiation process [4]. In view of the miscoding activity of negamycin, use of one species of radioactive amino acid in the assay of protein synthesis might not accurately reflect ribosomal activity. However, use of a mixture of 14C-labelled amino acids in the same system yielded similar results (data not shown). The miscoding activity of negamycin, like those of other miscoding antibiotics [7], is strongly dependent on Mg 2÷ concentration (data n o t shown), while its inhibition of the initiationfree protein synthesis was not, as shown above.

Negamycin prevents polysomes from disintegration The initiation-free protein synthesis proceeded with rapid disintegration of polysomes into single ribosomes, as shown in Fig. 3a. The disintegration was significantly retarded in the presence of 10 -3 M negamycin (Fig. 3b) where protein synthesis was inhibited by 44% (Table II). For comparison, the sedimentation profile of ribosomes after incubation with 6 • 10 -4 M chloramphenicol which inhibited protein synthesis by 88% is also shown (Fig. 3c, Table II). In a separate experiment performed under similar conditions, 2 • 10 -4 M streptomycin which inhibited protein synthesis by 46% (Table II) did n o t prevent polysomes from disintegration (data n o t shown), as reported previously [8]. In order to determine whether the polysome-freezing effect of negamycin is due to inhibition of the elongation and/or termination step, reaction mixtures were separated into the ribosome-bound peptide fraction and the released peptide fraction. As Table II shows, negamycin inhibited the release of peptides into the supernatant fraction b u t n o t significantly the accumulation of peptides in the ribosome-bound peptide fraction. The result was in contrast to those obtained with chloramphenicol and streptomycin, and suggests that negamycin binds to polysomal ribosomes and inhibits the termination process. Although

410

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Fig. 3. P r o t e c t i o n b y n e g a m y c i n o f p o l y s o m e s f r o m d i s i n t e g r a t i o n . Purified p o l y s o m c s (0.6 A 2 6 0 ) w e r e i n c u b a t e d w i t h a n d w i t h o u t n e g a m y c i n ( N G M ) or c h l o r a m p h e n i c o l (CM) at 3 7 ° C for t h e t i m e s i n d i c a t e d a n d a n a l y z e d in sucrose g r a d i e n t s as d e s c r i b e d in Materials a n d M e t h o d s . D o t t e d line r e p r e s e n t s t h e b a c k g r o u n d a b s o r p t i o n (sucrose g r a d i e n t a l o n e ) .

elongation was not affected appreciably, the process should undergo the miscoding effect of negamycin as it was the case in phage f2RNA-directed protein synthesis [4].

Bactericidal action of negamycin in the presence of chloramphenicol Negamycin, like other miscocting antibiotics, acts as a bactericidal agent [13]. However, it is distinct from the others in that its bactericidal action is not T A B L E II E F F E C T OF A N T I B I O T I C S ON T H E R E L E A S E OF PEPTIDES FROM RIBOSOMES R e a c t i o n m i x t u r e s , p r e p a r e d as d e s c r i b e d in t h e l e g e n d t o Fig. 3, c o n t a i n e d a n t i b i o t i c s w h e r e i n d i c a t e d . T h e m i x t u r e s w e r e i n c u b a t e d a t 3 7 ° C f o r i n d i c a t e d t i m e s a n d s u b m i t t e d t o s u c r o s e d e n s i t y g r a d i e n t cent r i f u g a t i o n as d e s c r i b e d in Materials a n d M e t h o d s . R a d i o a c t i v i t i e s l o c a l i z e d in t h e p o l y s o m e f r a c t i o n s , 70S r i b o s o m e f r a c t i o n s a n d t h e lighter f r a c t i o n s (all in h o t t r i c h l o r o a c e t i c a c i d - i n s o l u b l e m a t e r i a l s ) w e r e s u m m e d u p s e p a r a t e l y . SM, s t r e p t o m y c i n ; N G M , n e g a m y c i n ~ CM, c h l o r a m p h e n i c o l . Incubation time (rain)

Addition

[ 14C] Val incorporated (pmol)

D i s t r i b u t i o n o f [ 14C] Val i n c o r p o r a t e d ( p m o l ) Polysomes

Supernatant

Monosomes

r

II

3 20 20 20

-11.5 -25.0(100%) CM, 6 X 10 -4 M 3.0 ( 1 2 ) NGM, 10 -3 M 13.9 ( 5 6 )

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5.O 8.0(100%) 4.8 ( 6 0 ) 8.0 ( 1 0 0 )

18.7 28.6(100%) 15.6 ( 5 4 ) 22.9 ( 8 0 )

411

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0

1

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Fig. 4. E f f e c t of c h l o r a m p h e n i c o l and p u r o m y c i n o n the bactericidal effect of n e g a m y c i n . N e g a m y c i n ((a) at 2 • 1 0 -4 M; ( b ) a t 3 • 1 0 -4 M) and o t h e r antibiotics (CM, c h l o r a m p h e n i c o l a t 2 0 p g / m l ; PM, ptt~om y c i n a t 20 /~g/ml) w e r e a d d e d to e x p o n e n t i a l l y g r o w i n g E. cell B cells at times i n d i c a t e d (4). A t intervals, s a m p l e s w e r e t a k e n for c o u n t i n g viable cells as described in M a t e r i a l s a n d M e t h o d s .

diminished b y chloramphenicol nor stimulated by puromycin (Fig. 4). Among inhibitors of protein synthesis, the bactericidal action is mostly ascribed to irreversible effect of an agent on ribosomes [8,14]. Miscoding is n o t a direct cause of bactericidal action [15]. In this respect, one might remember that the bactericidal effect of streptomycin is masked in the presence of chloramphenicol [16] probably because chloramphenicol freezes polysomes to which streptomycin can n o t bind as strongly as to single ribosomes [8]. In view of the fact that negamycin inhibited the termination step in both phage f2RNA-directed protein synthesis [4] and the initiation-free protein synthesis and its bactericidal action was n o t diminished by chloramphenicol, it appears likely that negamycin binds irreversibly to polysomal ribosomes as to single ribosomes under physiological conditions. Discussion

It is desired to determine the primary effect of an antibiotic on living cells for physiological relevance. However, results are sometimes difficult to interpret because various processes are closely correlated with each other in living cells. As one step toward overcoming this problem, we used endogenous polysomes from E. cell to obtain some clues for elucidation of in vivo effect of negamycin. Normal termination in the initiation-free protein synthesis was supported by the observations that during the course of incubation, polysomes were converted into single ribosomes with a concomitant release of nascent peptides into the supernatant fraction (Fig. 3, Table II). However, radioactivity in the polysome and m o n o s o m e fractions of control run did n o t disappear completely even after a 20 min incubation. This may be due to some breakdown products of polysomes caused by contaminating ribonuclease. However, this system was suitable for analyzing the possible effect of negamycin on the termination process. The inhibition of termination by negamycin presented in

412 this paper contrasts the effect of streptomycin, spectinomycin and erythromycin which does not act on polysomal ribosomes as strongly as on single ribosomes [8,17,18]. Negamycin and streptomycin are similar in that they inhibited the initiationfree protein synthesis only partially, b u t they differ in that the former protected polysomes from disintegration while the latter enhanced the disintegration. Davis et al. [8] reported that this streptomycin effect was diminished by increasing the concentration of Mg 2÷. In contrast, the action of negamycin was n o t influenced significantly by Mg 2+ (Fig. 2). We have reported that inhibition of the termination reaction by negamycin was also insensitive to Mg 2÷ concentration as opposed to its miscoding activity [5]. Presumably codonanticodon recognition is strictly dependent on the conformation of ribosomes [7] while the negamycin-sensitive step of the termination process is not. These observations strongly support the conclusion that negamycin inhibits the termination step of the initiation-free protein synthesis. Assays of the effect of an antibiotic on living cells in the presence of another antibiotic whose mechanism of action is well known often provide useful information [ 16,19,20]. The bactericidal effect of negamycin was not diminished by chloramphenicol nor stimulated b y puromycin; chloramphenicol freezes polysomes [10] while puromycin enhances turn-over of the ribosome cycle [21] (Fig. 4). The simplest explanation of these observation would be that negamycin, unlike streptomycin and some other antibiotics, binds irreversibly to ribosomes at any stage of the r i b o s o m e cycle. Acknowledgements The authors wish to express their gratitude to Dr. Taiki Tamaoki Cancer Research Unit, McEachern Laboratory, University of Alberta, for criticism in preparing the manuscript. References 1 K o n d o , S., S h i b a h a x a , S., T a k a h a s h i , S., M a e d a , K., U m e z a w a , H. a n d O h n o , M. ( 1 9 7 1 ) J. A m e r . Chem. Soc. 93, 6305--6306 2 M i z u n o , S., N i t t a , K. a n d U m e z a w a , H. ( 1 9 7 0 ) J. A n t i b i o t . 2 3 , 5 8 9 - - 5 9 4 3 U e h a r a , Y., K o n d o , S., U m e z a w a , H., S u z u k a k e , K. a n d H o r i , M. { 1 9 7 2 ) J. A n t i b i o t . 2 5 , 6 8 5 - - 6 8 8 4 U e h a r a , Y., H o r i , M. a n d U m e z a w a , H. ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 3 7 4 , 8 2 - - 9 5 5 U e h a r a , Y., H o r i , M. a n d U m e z a w a , H. ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A c t a 4 4 2 , 2 5 1 - - 2 6 2 6 Tai, P.-C., Wallace, B . J . , H e r z o g , E . L . a n d Davis, B.D. ( 1 9 7 3 ) B i o c h e m i s t r y 1 2 , 6 0 9 - - 6 1 5 7 Davies, J., G i l b e r t , W. a n d G o r i n i , L. ( 1 9 6 4 ) P r o c . N a t l . A c a d . Sci. U.S. 5 1 , 8 8 3 - - 8 9 0 8 Wallace, B . J . , Tal, P.-C., H e r z o g , E . L . a n d Davis, B.D. ( 1 9 7 3 ) P r o c . N a t l . A c a d . Sci. U.S. 7 0 , 1 2 3 4 - 1237 9 Flessel, C.P., R a l p h , P. a n d R i c h , A. ( 1 9 6 7 ) S c i e n c e 1 5 8 , 6 5 8 - - 6 6 0 1 0 Flessel, C.P. ( 1 9 6 8 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 3 2 , 4 3 8 - - 4 4 6 11 O k u y a m a , A., M a c h i y a m a , N., K i n o s h i t a , T. a n d T a n a k a , N. ( 1 9 7 1 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 43, 196--199 1 2 Tai, P.-C., Wallace, B.J. a n d Davis, B.D. ( 1 9 7 3 ) B i o c h e m i s t r y 1 2 , 6 1 6 - - 6 2 0 1 3 M i z u n o , S., N i t t a , K. a n d U m e z a w a , H. ( 1 9 7 0 ) J. A n t i h i o t . 2 3 , 5 8 1 - - 5 8 8 1 4 G a r v i n , R . T . , Biswas, D . K . a n d G o r i n i , L. ( 1 9 7 4 ) P r o c . N a t l . A c a d . Sci. U.S. 7 1 , 3 8 1 4 - - 3 8 1 8 1 5 G o r i n i , L. a n d K a t a j a , E. ( 1 9 6 4 ) P r o c . N a t l . A c a d . Sci. U.S. 5 1 , 9 9 5 - - 1 0 0 1 1 6 P l o t z , P . H . a n d Davis, B . D . ( 1 9 6 2 ) J. B a c t e r i o l . 8 3 , 8 0 2 - - 8 0 5 17 Wallace, B . J . , Tai, P.-C. a n d Davis, B.D. ( 1 9 7 4 ) P r o c . N a t l . A c a d . Sci. U.S. 7 1 , 1 6 3 4 - - 1 6 3 8 1 8 Tal, P.-C., W a l l a c e , B.J. a n d Davis, B.D. ( 1 9 7 4 ) B i o c h e m i s t r y 1 3 , 4 6 5 3 - - 4 6 5 9 1 9 Y a m a k i , H. a n d T a n a k a , N. ( 1 9 6 3 ) J. A n t i h i o t . 1 6 , 2 2 2 - - 2 2 6 20 White, J.R. and White, H.L. (1964) Science 146, 772--774 21 R o n , E . Z . , K o h i e r , R . E . a n d Davis, B.D. ( 1 9 6 6 ) P r o c . N a t l . A c a d . Sci. U.S. 56, 4 7 1 - - 4 7 5