123

Biochimica et Biophysica Acta, 475 (1977) 123--130

© Elsevier/North-Holland Biomedical Press

BBA 98847 PEPTIDYL TRANSFERASE ACTIVITY IN WHEAT GERM RIBOSOMES EFFECT OF SOME ANTIBIOTICS

M.M. SIKORSKI a, j. (~ERN.~b, I. RYCHLfKb and A.B. LEGOCKI a,, a Institute of Biochemistry, Agriculture University 60-637 Pozna~, Wolynsha 35 (Poland) and b Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague 6, Flamingovo nam. 2 (Czechoslovakia)

(Received August 25th, 1976)

Summary The formation of N-acetyl-leucyl-puromycin in a "fragment.reaction" catalyzed by 80 S ribosomes from wheat germ was characterized. The reaction product was identified by high-voltage electrophoresis. The fragment reaction is inhibited by sparsomycin, blasticidin S, gougerotin and to a lesser degree by amicetin and tetracycline. Formation of an acLeu-pentanucleotide-ribosomes complex was strongly stimulated by sparsomycin.

Introduction Peptide bond formation is catalyzed by peptidyl transferase in a process involving the transfer of a nascent peptide chain from peptidyl-tRNA attached to the ribosomal P site to aminoacyl-tRNA bound to the A site. The nature of the peptidyl transfer reaction has been elucidated by means of the so-called "fragment reaction" in which a terminal fragment of peptidyl or N-acyl-aminoacyl-tRNA functions as a model of the donor substrate [1]. Bacterial and mammalian peptidyl transferases have been relatively well characterized [2--7]. However, no systematic studies have so far been reported on the enzyme from higher plants. In the present paper, we describe the properties of wheat germ peptidyl transferase with the aid of a fragment system with C-A-C-C-A-ac-[ 14C]Leu as a donor substrate. Materials and Methods Materials

Puromycin dihydrochloride was obtained from Nutritional Biochemicals Co., * To whom correspondence should be add~ssed.

124 amicetin from Bristol, U.S.A., gougerotin from Calbiochem, U.S.A., sparsomycin from Upjohn, U.S.A., tetracycline from Spofa, Czechoslovakia, blasticidin S was a gift from Professor Lichtenthaler, Technische Hochschule, Darmstadt, G.F.R., L-[14C]leucine, 126 Ci/mol was from the Institute for Research, Production and Application of Radioisotopes, Czechoslovakia; t R N A Escherichia coli was kindly provided by Dr. J. Jonak from the Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences. Wheat germ ribosomes were prepared and purified as described previously [8]. Ac- [ ~4C] Leu-tRNA was prepared according to Haenni and Chapeville [9].

Preparation of ac-[14C]Leu-pentanucleotides C-A-C-C-A-ac-[14C]Leu and U-A-C-C-A-ac-[14C]Leu were prepared from ac-[~4C]Leu-tRNA b y digestion with ribonuclease T~, Calbiochem, U.S.A. Usually, 20 mg of ac-[14C]Leu-tRNA were digested with 100 pg RNAase in 1.5% sodium acetate pH 5.4 containing 0.8 mM EDTA for 60 min at 37 ° C. The terminal fragments were isolated by high-voltage electrophoresis on Whatman 3 MM paper in buffer containing 0.5% pyridine/5% acetic acid, pH 3.5 for 5 h at 50 V/cm according to Monro et al. [1].

Transfer assay The reaction of ac-[14C]Leu-pentanucleotide with puromycin was carried o u t according to the conditions of the fragment reaction described by Monro et al. [10]. The complete reaction mixture contained 170 pg ribosomes, ac-[ ~4C]Leu-pentanucleotide, 2000 cpm, 1 mM puromycin, 50 mM Tris • HC1 buffer pH 7.6, 350 mM KC1 and 15 mM magnesium acetate in a total volume of 100 pl. The reaction was initiated by the addition of 50 pl methanol, and incubation was carried o u t for 2 h at 0°C. The reaction was terminated with 100 pl 0.1 M sodium acetate, pH 5.5 in a saturated solution of magnesium sulphate [11]. The reaction product was then extracted into 1.5 ml ethyl acetate. Samples of 1.2 ml were mixed with 5 ml of Bray scintillation fluid and counted in a Packard scintillation spectrometer. The a m o u n t of ac-[14C]Leu-residues transferred from the ac-[~4C]Leu-pentanucleotide to the acceptor ( p u r o m y c i n ) w a s estimated as the difference b e t w e e n the radioactivity extracted into the organic solvent after incubation with and w i t h o u t puromycin.

Identification of the reaction products The products formed in the transfer reaction were extracted into ethyl acetate and identified by high-voltage electrophoresis on Whatman 52 paper according to Prusik et al. [12].

Binding of the donor substrate The binding of C-A-C-C-A-ac-[14C]Leu to ribosomes was assayed according to Monro et al. [13]. The reaction mixture contained per 100 pl: 280 pg ribosomes, 50 mM Tris • HC1 buffer pH 7.6, 350 mM KC1, 15 mM magnesium acetate and ac-[14C]Leu-pentanucleotide, a b o u t 1000 cpm. The reaction was initiated b y addition of 50 pl ethanol and incubation was carried out at 0°C. After a 2-h incubation ribosomes were centrifuged and aliquots of supernatant were counted in a Bray scintillator. The a m o u n t of ac-[14C]Leu-pentanucleo -

125 tide bound to ribosomes was determined from the difference between the radioactivity present in the incubation mixture without ribosomes and that remaining in the supernatant after binding reaction, and expressed as pmol of donor substrate bound. Results

Wheat germ ribosomes catalyse peptide bond formation under the conditions of the fragment reaction, CACCA-ac-[14C]Leu as a donor substrate and with puromycin as acceptor substrate [ 10]. Fig. 1 shows the time course of the fragment reaction with acLeu-pentanucleotide and puromycin catalysed by wheat ribosomes. The rate of the fragm e n t reaction is proportional to the incubation time and the reaction is not completed even after 3 h incubation. In the absence of puromycin there was a very small a m o u n t of radioactivity extracted into ethyl acetate. The fragment reaction with wheat germ ribosomes depends on the presence of K ÷, Mg2÷ and methanol (Fig. 2A, 2B, 2C). The optimum concentrations for both cations are 350 mM for K ÷ and 15 mM for Mg 2÷. The dependence of the reaction on methanol is similar to that observed with ribosomes from E. coli and mammals. The reaction is optimal at 33% of methanol. It is seen from Fig. 2D, that the transfer of acLeu-residue to puromycin is proportional to the ribosome concentration up to 280 pg of ribosomes per 100 pl of reaction mixture. The products of the transfer reaction were identified by high-voltage paper electrophoresis. Fig. 3B shows that, at pH 3.5, about 90% of the radioactivity migrates as ac-[14C]Leu-puromycin. A slowly migrating minor component, also present in the control run in the absence of puromycin (Fig. 3A), may be a degradation product.

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Fig. 2. G e n e r a l r e q u i r e m e n t s of t h e f r a g m e n t r e a c t i o n in t h e w h e a t g e r m s y s t e m , (A), e f f e c t of K + c o n c e n t r a t i o n ; (B), e f f e c t o f Mg 2+ c o n c e n t r a t i o n ; (C), e f f e c t of m e t h a n o l c o n c e n t r a t i o n ; (D), e f f e c t o f r i b o s o m e s c o n c e n t r a t i o n . I n c u b a t i o n was c a r r i e d o u t a t 0 ° C f o r 2 h in t h e c o n d i t i o n s d e s c r i b e d in Materials a n d Methods. The values were corrected for the minus p u r o m y c i n blanks.

TABLE

I

ACTIVITY

O F C-A-C-C-A-ac-[ 14C] L e u - R I B O S O M E

COMPLEX

IN T H E

FRAGMENT

REACTION

T h e standard incubation mixture contained, before alcohol addition: 50 m M Tris • H C I p H 7.6, 15 m M m a g n e s i u m acetate, 3 5 0 m M KCI, ac-[14C]Leu-pentanucleotide (2000 cpm), 2 0 0 ~g ribosomes and, where indicated, 1 0 /aM sparsomycin and 1 m M p u r o m y c i n in a total v o l u m e of 100/~l. T w o sets of tubes were incubated in parallel: the first set was tested for binding of acLeu-fragment to ribosomes, whereas, the second one for the acLeu-puromycin formation. Expt.

Incubation time (rain)

Additions 0 rain

60 rain

acLeu-fragment b o u n d to ribosomes (pmol)

acLeu-puromycin formed (pmol)

1

120

puromycin

--

1.85

1.61

2

120

sparsomycin

--

3.90

0

3

120

p u r o m y c i n plus sparsomycin

--

3.08

0.28

4

120

puromycin

sparsomycin

2.72

0.72

5

120

sparsomycin

puromycin

3.70

0.20

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128

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Anti,biotcs (tog M) Fig. 5. E f f e c t o f v a r i o u s a n t i b i o t i c s o n f r a g m e n t r e a c t i o n w i t h w h e a t g e r m r i b o s o m e s . A s s a y s c a r r i e d o u t in s t a n d a r d s y s t e m c o n t a i n i n g t h e a n t i b i o t i c s a t a c o n c e n t r a t i o n i n d i c a t e d . 1, s p a r s o m y c i n ; 2, b l a s t i c i d i n S; 3, g o u g e r o t i n ; 4, t e t r a c y c l i n e ; 5, a m i c e t i n .

As in the bacterial system [13], sparsomycin induces in the wheat germ system the formation of an inert complex between the 50 S subunit and the CCA-peptidyl moiety [13]. Sparsomycin stimulates ribosomal binding of Nacetyl-leucyl-pentanucleotide in the presence of alcohol (Fig. 4). This stimulation increased with increasing antibiotic concentration, and a maximum stimulation, 2-fold was observed at 10 -s M concentration. The relation between the sparsomycin induced formation of an inert complex and fragment reaction in wheat germ system is shown in Table I. Incubation of ribosomes with acLeu-pentanucleotide in the presence of puromycin alone led to acLeu-puromycin synthesis (Expt. 1, Table I), while incubation with sparsomycin alone led to complex formation (Expt. 2). When puromycin and sparsomycin were added simultaneously, the larger part of the substrate reacted with sparsomycin and only a smaller part with puromycin (Expt. 3). Preincubation with puromycin lowered the extent of sparsomycin-induced complex formation (Expt. 4). The sparsomycin induced complex, once formed, did not react with puromycin (Expt. 5). According to these results sparsomycin inhibits the puromycin reaction by inducing the formation of an inert complex as in the bacterial system [13]. Fig. 5 compares the effects of various antibiotic inhibitors of protein synthesis on the fragment system. All the antibiotics assayed, sparsomycin, blasticidin S, gougerotin, tetracycline and amicetin, were effective, which confirms their known specificity with regards to peptide bond formation in different species [5,7,14]. Sparsomycin, blasticidin S and gougerotin were more effective than tetracycline and amicetin in the reaction tested. Discussion

It is already well established that peptide bond formation involves an enzyme peptidyl transferase, an integral part of the larger ribosomal subunit.

129

The mode of action of peptidyl transferase seems to be very similar in prokaryotic and eukaryotic ribosomal systems as revealed by a comparison of the properties of peptidyl transferase from E. coli [2] yeast [3], human tonsils [5] and rat liver [7]. In this report, these studies are extended to a plant system. As in the previous communications peptidyl transferase is investigated by using the fragment reaction which represents peptide bond formation uncoupled from other reactions of protein synthesis. Our results show that requirements and properties of wheat germ peptidyl transferase correspond to those described earlier for the analogous enzymes of various origin, although some differences observed may be characteristic of the plant enzyme. Thus, the kinetics of transfer of AcLeu-residue from C-A-C-C-AacLeu catalyzed by wheat germ ribosomes differs from that of the reactions catalyzed by bacterial and mammalian ribosomes. The transfer reaction continues for more than 3 h, whereas in the bacterial and mammalian case the reaction stops after 10--30 min. The transfer reaction, likewise other ribosomal processes, requires the presence of inorganic ions. The optimal concentration of K ÷ and Mg~+ for the fragment reaction was 350 mM and 15 mM, respectively. Similarly, as in other systems described earlier [1,3,5,7,10], the fragment reaction with wheat germ ribosomes is also dependent upon the presence of alcohol which is considered to promote interaction between a short donor fragment and ribosomes [10]. Much information on the mechanism of peptide bond formation has been gained by the use of specific antibiotics. Sparsomycin, blasticidin S, gougerotin, tetracycline, and amicetin proved to be effective inhibitors of the fragment reaction in our system of plant 80S ribosomes from wheat germ. As in the bacterial system [13], in the presence of 33% methanol, 10 -s M sparsomycin stimulated binding of the C-A-C-C-A-acLeu fragment to ribosomes by more than 200%. Preincubation of the system with puromycin lowers the yield of sparsomycin-induced complex. When the wheat ribosomes were incubated with puromycin and sparsomycin simultaneously, the main product was identified as the acLeu-fragment-ribosome complex and only a small amount of acLeu-puromycin was formed. These results show that sparsomycin-induced binding of acLeu-fragment to wheat ribosomes leads to a stable complex, which does not seem to react with puromycin. The present results represent a preliminary stage in the study of peptidyl transferase in the system of plant ribosomes. Acknowledgements This work was supported by the Polish Academy of Sciences within the project No. 09.7.-1.2.7. References 1 Monro, R.E., Cernd, J. and Marcker, K.A. (1968) Proc. Natl. Acad. Sci. U,S. 61, 1042--1049 2 Monro, R.E., Staehelin, T., Celma, M.L. and Vazquez, D. (1969) Cold Spring Harbor Syrup. Quant. Biol. 34, 357--366 3 Vazquez, D., Battaner, E., Neth, R., HeHer, G. and Monro, R.E. (1969) Cold S ~ H m d ~ Syrup. Quant. Biol. 34, 369--375

130 4 Rychlfk , I., Cern~, J., Cb_l~dek, S., Pulkr~ibek, P. and Zemli~ka, J. (1970) Eur. J. Biochem. 1 6 , 1 3 6 - 142 5 Neth, R., Mortro, R.E., Hener, G., Battanez, E. and Vazquez, D. (1970) FEBS Lett. 6 , 1 9 8 - - 2 0 2 6 Caskey, C.T., Beaudet, A.L., Scolnick, E.M. and Rosman, M. (1971) Proc. Natl. Acad. Sei. U.S. 68, 3163--3167 7 Irmanen, V.T. and NichoUs, D.M. (1974) Biochim. Biophys. Acta 3 6 1 , 2 2 1 - - 2 2 9 8 GoliSska, B. and Legocki, A.B. (1973) Biochim. Biophys, Aeta 324, 156--170 9 Haenni, A.L. and Chapeville, F. (1966) Biochim. Biophys. Acta 114, 135--148 10 Monro, R.E. and Marcker, K.A. (1967) J. Mol. Biol. 2 5 , 3 4 7 11 Cern~, J., R y c h l l k , I., Zemlfcka, J. and Cb-l~dek, S. (1970) Biochim. Biophys. Acta 204, 203--209 12 Prusfk, Z. and Keil, B. (1960) Collection Czech. Chem. Commun. 25, 2 0 4 9 - - 2 0 5 4 13 Monro, R.E., Celma, M.L. and Vazquez~ D. (1969) Nature 2 2 2 , 3 5 6 - - 3 5 8 14 Cern~, J. R y c h l f k , I. and Lichtenthaler, F.W. (1973) FEBS Lett. 30, 147--150