Eur. J. Biocheni. 58, 397-402 (1975)

The Photochemical Inactivation of Peptidyl Transferase Activity Kwong K. WAN, Nasir D. ZAHID, and Ross M . BAXTER Faculty of Pharmacy, University of Toronto (Rcccived May 2, 1975)

The photochemical oxidation of the 5 0 3 ribosomal subunit results in a rapid irreversible loss of peptidyl transferase activity. The first-order rate of inactivation occurring during the first forty minutes suggests that a single reactive group is being inactivated. The pH profile of inactivation exhibits a maximum at pH 7.5. Erythromycin at a low concentration (0.04 pmol) affords significant protection. Puromycin also exerts a protective effect but at higher concentrations. Chloramphenicol, sparsomycin and lincomycin did not exert a protective effect. The loss in catalytic activity was not accompanied by a loss in substrate binding affinity of the donor and acceptor substrates.

Photo-oxidation in the presence of the anionic dye Rose Bengal effectively modifies proteins under very mild conditions and exhibits under these conditions a high degree of specificity for histidine and guanine [I]. Garvin et al. [2] have reported inactivation of Escherichiu coli 70-S ribosomes by photo-oxidation in the presence of Rose Bengal and other sensitizing dyes. Noller ef al. [3] have shown that photo-oxidation of 30-S ribosomal subunits in the presence of the anionic dye Rose Bengal leads to a rapid loss of activity in protein synthesis and that three proteins and about three histidine residues are protected from photooxidation by the presence of poly(U) and tRNA. Affinity analogues of peptidyl-tRNA [4] and chloramphenicol [5,6] have provided evidence that the proteins L16, L2, L26-27 are located at the peptidyl transferase center and are probably closely associated with each other. We report here the dye-sensitized photoinactivation of peptidyl transferase activity, some characteristics of the inactivation and the protection of peptidyl transferase activity by erythromycin and puromycin but not by sparsomycin, lincomycin or chloramphenicol. MATERIALS AND METHODS Muter ials

~-[U-'~C]Phenylalanine, ~ - [ u - ' ~ c ] L e u - t R Nand A ~ - [ u - ' ~ c ] P h e - t R N were A obtained from New England Nuclear. Sparsomycin was a generous gift from Dr Alfred R. Stanley (National Institutes of Health) and lincomycin was a generous gift from the Upjohn

Company. Puromycin dihydrochloride (neutralized with Tris base before use) was obtained from Nutritional Biochemicals Corporation. Rose Bengal was obtained from Fisher Scientific Company (examined for homogeneity by paper chromatography and electrophoresis). Buffer solutions were as follows. Buffer I : 10 mM Tris-HC1 (pH 7.5); 10 mM MgCI2; 30 mM NH4Cl; 0.5 mM dithiothreitol and 0.5 mM EDTA. Buffer I1 : 10mM Tris-HC1 (pH7.5); 5 m M MgCI,; 200mM NH4Cl and 0.25 mM EDTA. Buffer 111: 33 mM TrisHCl (pH 7.8); 133 mM MgCI, and 267 mM KCI. Preparation of Ribosomes

Ribosomes from E. coli MRE 600 were prepared as described by Traub et al. [7] and Staehelin and Maglott [S]. The ribosomal pellet was resuspended in buffer I. 50-S ribosomal subunits were prepared by dissociation from 70-S ribosomes and by dialysis against a buffer with a low concentration of Mg2+ and the 50-S ribosomal subunits were isolated according to Staehelin and Maglott [8] and stored in buffer 11. The 5 0 3 subunits so isolated had less than 5 contamination with 30-S subunits (as determined by analytical centrifugation). Preparation of Donor Substrates

AC-L-[U-14C]Phe-tRNA and Ac-L-[U-14C]LeutRNA were prepared according to the method of Haenni and Chapeville [9]. The products were divided

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into small aliquots, lyophilized and stored in an evacuated desiccator at 4 "C. The extent of acetylation was determined by the method of Schofield and Zamencnik [lo]. The specific activities were estimated 384 Ci/mol and AC-Las : Ac-~-[u-~~c]Phe-tRNA [U-14C]Leu-tRNA 254 Ci/mol.

Photochemical Inactivation of Peptidyl Transferase Activity

and then centrifuged at 4000 x g for 10 min at 2- 4 "C. The supernatants (0.1 ml) were removed and counted as before. Parallel incubations without ribosomes were done for the estimation of the total radioactivity under identical conditions. The amount of bound substrate was calculated by difference. Duplicates of experiments were performed and the results were averaged.

Assay of Peptidyl Transferase Activity

The assay is a modification of the "fragment reaction" developed by Monro and Marcker [12]. It was carried out essentially according to Miskin et al. [ll]. A standard reaction mixture contained (unless otherwise specified): 70-S ribosome or its larger subunit (50-s), 5 ,4260 units; Ac-~-[u-l~ClPhe-tRNA or 1000- 4000 counts/min ; Ac-L-[U-'~C]L~U-~RNA, neutralized puromycin, 0.8 mM ; and 33 % CH30H in a total volume of 150 pl of fragment buffer. Before initiation of reaction, the ribosomes were preincubated in the buffer alone at 40°C for 5 min to ensure that most of the ribosomes were in the active state [13,14]. Incubations were carried out in small conical tubes at 25 "C for 15 to 20 min or a longer time when indicated. The reactions were stopped by adding 10 yl of 1 0 N KOH to each tube and then incubated at 40°C for 5 min, after which 1 ml of 1 M potassium phosphate buffer (pH 7.0) was added. To estimate the extent of reaction, the above mixture was extracted with 2 ml of ethylacetate [15,16] by shaking in a Vortex tube mixer for 5 to 10 s and then centrifuged briefly at 2000 rev./min (IEC model CS). Over 95 % of the N-Ac-~-[U-'~C]aminoacylpuromycin was recovered in the ethylacetate layer and 1.5 ml was then removed and counted in a Packard Tricarb liquid scintillation spectrophotometer using toluene/PPO/ POPOP/Triton X-100 as the medium. The average counting efficiency was 80 to 90%. Each experiment was done in duplicate and the average values were tabulated. Extent of reaction was usually expressed as percentage of acylaminoacyl-tRNA converted to acetylaminoacylpuromycin. Unless otherwise stated, a background value of 60 - 90 counts/min obtained without puromycin was subtracted from values obtained with puromycin.

Binding of L-[ U-'4C]Phe-tRNA to Ribosomes

Substrate and antibiotic interactions at the A site on the peptidyl transferase center were assayed according to Harris and Pestka[20], Celma et al. [21] and Vogel et al. [19] with minor alterations. Each 0.15-ml reaction mixture contained (unless otherwise , 7.54 pmol specified): ~ - [ u - l ~ C l P h e - t R N A0.95(800- 7000 counts/min, ribosomes, 10 A260 units; and ethanol, 33%; in an ionic environment identical to fragment buffer. Reaction mixtures were incubated at 24°C for 60 min to achieve equilibrium binding. Reactions were stopped by chilling in ice and then centrifuged immediately at 4000 x g for 10 min at about 2 - 4 "C. Extent of binding was determined as before in the binding of substrates to P site. Binding Using [3H]Puromycin

For the binding of [3H]puromycin, samples containing 5 A260 units of 50-S ribosomal subunits suspended in 0.1 ml of buffer I11 were put into dialysis bags, which were pretreated with sodium bicarbonate and soaked in buffer I11 overnight, and then dialysed against 3.1 ml of the same buffer containing 872938 counts/min of [3H]puromycin, with or without chloramphenicol for 24 h at 4- 8 "C with shaking. Extent of binding was obtained by noting the difference between the counts inside and outside of the bag by counting 50-yl aliquots from each compartment. Equilibrium dialysis under the same conditions except in the absence of ribosomes was carried out and counts on both sides of the bag were found to be the same. Photochemical Modification of Ribosomes

Photo-oxidation of the 50-S ribosomal subunits was carried out similar to Noller et al. [3] and Garvin et al. [2]. The reaction mixture (0.24 ml) contained Assay of substrate and antibiotic interactions at 10A2,, units of 50-S ribosomal subunits in a total the P site on the peptidyl transferase center was carried volume of 0.24 ml of buffer (33 mM Tris-HC1, pH 7.5; out according to Celma et al. [18] and Vogel [19]. 13 mM MgC12; 267 mM KC1) and 0.1 pmol of Rose The standard incubation mixture contained (unless Bengal. The reaction vessels (small 2-ml glass vials otherwise specified):7 0 3 ribosomes or 50-S ribosomal subunits ( 5 - 10 ,4260 units); N-Ac-~-[u-'~ClPhe- wrapped with aluminium foil) were placed in an ice bath. Before reaction, the mixture was shaken gently tRNA, 2000- 3000 counts/min; and 33- 50% preat 0 "C for a few seconds to ensure maximum amount cooled ethanol as indicated in separate experiments, of air dissolving in it. It was then irradiated with a in a total volume of 150 pl of fragment buffer. Aliquots 150 W Westinghouse reflector spot light held 15 cm were incubated in small test tubes at 0°C for 45 min Binding of N-Acetylaminoacyl-tRNA to Ribosomes

K. K. Wan, N. D. Zahid, and R. M. Baxter

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0.1 Amount of dye ((Lmd)

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Fig. 1. Inactivation of’ SO-S ribosomal peptidyl transjerase activity by different concentrations of dye (Rose Bengal). 50-S subunits were photo-oxidized for 50 min and then assayed for peptidyl transferase activity as described in Materials and Methods. (I) -@ (. ) photo-oxidation in presence of various concentrations of dye; (11) (A-A) subunits incubated with the same amount of dye as in (I) except without exposure to irradiation

directly above for a specified period of time. The reaction was kept on ice throughout the course of photo-oxidation. When the reaction was terminated, the mixture was either directly assayed for activity (see Results) or the modified ribosomes were precipitated by 0.65 volume of ethanol, as described by Staehelin et al. [22], and resuspended in buffer (the activities in both cases were found to be the same). Ribosomes were also collected by centrifugation. All steps mentioned above were carried out in the dark.

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50

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Time (min)

Fig. 2. Rate of inactivation by photochemical oxidation. Ten A,,, units of SO-S ribosomal subunits in a total volume of 0.24 ml were photo-oxidized, as described in Materials and Methods. At the times indicated, vials were withdrawn, and the contents were diluted to 1.0 ml with buffer 111, precipitated with 0.65 ml of cold ethanol and resuspended in 0.24 ml of a buffer containing 100 mM Tris-HC1 (pH 7.8), 40 mM MgCI, and 800 mM KCI. Portions (60-p1aliquots containing 2.3 A,,,, units of ribosomes were used for peptidyl transferase assay as described in Materials and Methods. (I) (M inactivation ) by photo-oxidation in presence of Rose control ) Bengal; (11) (H

-

\

kbz0.0805 miti‘

RESULTS Efect of Dye Concentration and Rate

Photo-oxidation of the 50-S ribosomal subunit results in a rapid loss of peptidyl transferase activity. The effect of the time of photo-oxidation and the concentration of dye (Rose Bengal) on the peptidyl transferase activity are demonstrated in Fig. 1 and 2. It will be observed that the inactivation progressed with time. Actually after sixty minutes more than 95 % inactivation was achieved. The appropriate controls, namely photo-oxidation of 50-S subunits without dye and the dark reaction in which 50-Ssubunits incubated with dye in absence of light indicated that the progressive loss of peptidyl transferase activity was indeed due to photo-oxidation. A semilog plot of percentage activity remaining against time (values as plotted in curve I, Fig.2) yielded a biphasic curve (Fig.3). The first phase of the inactivation process, corresponding to 0 to 40 min is a straight line indicating a first-order rate process for the inactivation of peptidyl transferase activity with a rate constant, k, of 0.0347 min-’. The second phase (40 to 60 min) of inactivation is also a straight

86. 4 .

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Fig.3. A semilog plot of the percentuRe uciivity remaining versus time of the inactivation of peptidyl trun.ferase activity by photooxidution in presence of Rose Bengul

line ; however, the rate constant corresponding to this time period in the inactivation process is different from the first phase (kb = 0.0805 min-I). Such evidence is suggestive that the complete loss of activity may involve the inactivation of two different but specific groups. In light of the observed effect all subsequent results refer to a forty-minute period of photo-oxidation. Effect of p H on Inactivation

The specificity of photo-oxidation has been shown [23- 251 to be affected by and dependent on pH since pH affects not only protein conformation but also the

Photochemical Inactivation of Peptidyl Transferase Activity

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Fig. 4.p H profile of thr photochrmicul inactivution of"-S ribosomul peptidyl transferase activity. Ten A,,, units of 5 0 3 ribosomal subunits suspended in a total volume of 0.24 rnl of buffer, having the pH valuc as indicated, were photo-oxidized in presence of 0.1 pmol

of Rose Bengal for 30min under the conditions described in Matcrials and Methods. At the end of the reaction, the mixture was processed as described in Fig. 2. The percentage inhibition was plotted a s shown, against pH. (W) Citrate phosphate; (A)phosphate; (0)Tris-HCI

oxidation rate of certain amino acids. For example, the oxidation of histidine or histidine in protein has been shown to be pH-dependent with the protonated form being resistant to oxidation. A pH profile for the photoinactivation of peptidyl transferase activity is shown in Fig. 4.

Effects of Antibiotics on the Inuctivution of Prptidyl Transferase A number of antibotics which inhibit protein synthesis in intact cells or cell-free systems exert their effects by inhibiting peptide bond formation of the peptidyl transferase centre [26]. A number of these antibiotics were tested for their ability to protect peptidyl transferase against photochemical inactivation. Fig. 5 illustrates the protective effect of erythromycin. In contrast to erythromycin, chloramphenicol, sparsomycin and lincomycin did not exert a protective effect at the same or higher concentrations. It has been observed (unpublished data) when erythromycin was exposed to photo-oxidation conditions no inhibitory product was produced, and when present in the assay system in the concentrations utilized no inhibition was produced by erythromycin or Rose Bengal.

Binding Activities of the Modified 50-S Ribosomal Subunits A number of antibiotics may bind at or near the peptidyl transferase centre [26] thus affecting peptide

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Fig. 5 . t c e ' j E of erythromycin on the inactivation ofprpri+l transjerase by photochemical oxidation. Conditions: (I) ( 0 4 )as in Fig.2. (11) (r-V) (I) plus 0.04 pmol erythromycin in 0.24 ml of reaction mixture. (111) Control

bond formation and the binding of substrates to the donor and/or acceptor sites. To test for substrate interactions with native and modified 50-S ribosomal subunits, Ac-~-[u-'~c]Phe-tRNAor Ac-L-[U-'~C]Leu-tRNA was used as the donor substrate and ~ - [ u - ' ~ c ] P h e - t R N or A puromycin was used as the acceptor substrate. These were allowed to bind to ribosomal subunits non-enzymatically in the absence or presence of antibiotics under conditions identical or close to that used in the assay for peptidyl transferase activity. The results are presented in Table 1. It is evident from these results that although peptidyl transferase activity was lost the dye-sensitized photo-oxidation did not affect the ability of the 50-S ribosomal subunits to bind the donor and acceptor substrates. The binding of the donor was stimulated by erythromycin and sparsomycin but inhibited by lincomycin in both the normal and modified 50-S subunits a fact consistent with previous reports [21,22]. When [3H]puromycin was used as the acceptor substrate both the native and modified ribosomal subunits exhibited the same order of activity and the binding was inhibited by chloramphenicol as has been reported [28] although the inhibitions was reduced in the case of the modified 50-S ribosomal subunits.

Photo-Oxidation and Sulphydryl Groups Among the amino acids affected by dye-sensitized photo-oxidation are cysteine [23,24]. Although - SH groups appear not to be involved in peptide bond formation [29,30] they are important in subunit interactions. As shown in Table 2, 2-mercaptoethanol did not prevent inactivation of peptidyl transferase ac-

K. K. Wan, N. D. Zahid, and R. M. Baxter

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Table 1. Interaction of substrates with photo-oxidized 50-S ribosomal subunit The 50-S subunits were photo-oxidized as dcscribed in Materials and Methods. The binding of donor and acceptor substrates were carried out also as dcscribed in Materials and Methods. The concentration of antibiotic used was as shown in the table and rcpresented the final concentration Subslrdte

Antibiotic

Binding activity control

counts/min (a) DcJnorsubstrate (P site) Ac-I.-[U-'~C]P~~-~RNA

photo-oxidized

(x)

photo-oxidized control 0

i

/o

none sparsomycin 0.16 mM erythromycin 0.16 mM lincomycin 0.10 mM chlorainphenicolO.10 mM

1764 2240 2187 1282 2080

(100) (127) (124) (73) (1 18)

1640 2164 2935 1201 1410

(100) (132) (179) (73.5) (86)

93

none sparsomycin 0.1 mM erythromycin 1 mM lincomycin 0.1 mM chloramphenicol 1 mM

1012 (100) 1052 (104) 911 (90) 1035 (102) 1062 (105)

974 935 867 1150 1003

(100) (96) (89) (118) (103)

96

none chloramphenicol 0.5 mM

3278 (100) 1494 (46)

3241 (100) 2629 (81)

(b) Acceptor substrate (A site) ~ - [ u - l ~ C l P h e- t R N A

[3H]Puromycin

Table 2. Irreversihle inhibition of peptidyl transferase activity by dye-semitized photo-oxidation The 50-S ribosomal subunits were photo-oxidized as described in Materials and Methods for 50 min either with or without 2-mercaptoethanol (2-ME). Portions of the reaction mixtures were subjected to exhaustive dialysis against 600 volumes of buffer 111 containing 10 mM 2-mercaptoethanol for 48 h with constant stirring in the cold. Aliquots of the preparations, before and after dialysis, were assayed for peptidyl transferase activity as in Fig. 2 Experiment

Activity before dialysis after dialysis

% Normal reaction 5 0 4 + irradiation (control) SO-S dye irradiation 50-S dye + irradiation 10 mM 2-mercaptoethanol

+

+ +

+

100 98 10

100 75 2

15

10

tivity during photo-oxidation nor did its presence in 10 mM concentration in the dialyzing buffer result in any regeneration of activity. Thus it seems unlikely that -SH groups are involved in this inactivation of peptidyl transferase.

Irreversible Effect of Photo-Oxidation The irreversible nature of the action of photochemical oxidation is indicated in Table 2.

98.9

DISCUSSION When 50-S ribosomal subunits from E. coli MRE 600 were subjected to photo-oxidation in the presence of Rose Bengal there was a rapid loss of peptidyl transferase activity. The presence of the dye without exposure to light or exposure to light in the absence of dye produced no loss of this activity (Fig. 1). This loss of activity is attributed to the specific inhibition of peptidyl transferase and may be due to the preferential inactivation of a single reactive group in that part of the ribosome structure responsible for peptidyl transferase activity. This is indicated by the first-order rate of inactivation of activity occurring during the first forty minutes of the photochemical oxidation (Fig. 3). As shown in Fig.4 the photo-inactivation of the peptidyl transferase activity was pH dependent and has a maximum around pH 7.5. A similar pH dependence has been observed for peptidyl transferase activity in the formation of N-formyl-methionylpuromycin in the fragment reaction [29]. The pH profile of inactivation is indicative of a group ionizing around neutrality and the involvement of a histidine residue is suggestive. The protective effect of erythromycin (Fig. 5 ) and to a lesser extent puromycin and the lack of protection by chloramphenicol, sparsomycin and lincomycin tends to indicate a specific site of inactivation in peptityl transferase. The low concentration (0.04 pmole) of erythromycin which affords protection further supports the relatively unique protection

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K. K. Wan, N. D. Zahid, and R. M. Baxter: Photochemical Inactivation of Peptidyl Transferase Activity

exerted by erythromycin against inactivation by photochemical inactivation. The loss in catalytic activity by peptidyl transferase was not accompanied by a loss in the substrate binding affinity of 50-S ribosomal subunits for donor and acceptor substrates as evidenced from the data in Table 1. The binding of donor was stimulated by erythromycin and sparsomycin but inhibited by lincomycin in both the normal and modified 50-S subunits an observation consistent with those reported previously [21,22]. When [3H]puromycin was used as the acceptor substrate the results for both the native and modified 50-S subunits were consistent with that previously reported [28]. In conclusion the loss of peptidyl transferase activity as the result of photochemical oxidation of 50-S ribosomal subunits appears to represent a relatively selective irreversible inactivation of this activity. The selective protection observed with erythromycin and to a more limited extent puromycin is in contrast to the lack of protection by chloramphenicol, sparsomycin and lincomycin lends to support to this assumption. The retention of the binding capacity of the modified 50-S subunits for donor and acceptor substrates indicates a loss in the catalytic activity of peptidyl transferase as the only measurable effect observed under these experimental conditions. Further work is in progress to ascertain which of the 50-S ribosomal protein(s) have been modified. REFERENCES 1. Westhead, E. W. (1965) Biochemistry, 4 , 2139-2144. 2. Garvin, R . T., Julian, G. R. & Rodgers, S. J. (1969) Science (Wash. D . C . ) 164, 583-584. 3. Noller, H. F., Chang, C., Thomas, G. & Aldridge, J . (1971) J . Mol. Biol. 61, 669- 679.

4. Eilat, D., Pellegrini, M., Oen, H., De Groot, N., Lapidot, Y. & Cantor, C. R. (1974) Nature (Lond.) 250, 514-516. 5. Sonenberg, M., Wilchek, M. & Zamir, A. (1973) Proc. Nut1 Acad. Sci. U.S.A. 70, 1423-1426. 6. Pongs, O., Bald, R. & Erdmann, V. A. (1973) Proc. Nail Acud. Sci.U.S.A. 70,2229-2233. 7. Traub, P., Mizushima, S., Jowry, C. Y. & Nomura, M. (1971) Methods Enzymol. 20 C, 391 -407. 8. Staehelin, Y . & Maglott, D. R. (1971) Meth0d.s Enzymol. 20C. 449 -456. 9. Haenni, A. L. & Chapeville, F. (1966) Biochem. Biophys. Acta, 114, 135-148. 10. Schofield, P. & Zamencnik, P. C. (1968) Biochem. Biophys. Acts, 155, 410-416. 11. Miskin, R., Zamir, A. & Elson, D. (1970) J . Mol. Biol. 54, 355- 378. 12. Monro, R. E. (1967) J . Mol. Biol. 26, 147-151. 13. Miskin, R., Zamir, A. & Elson, D. (1968) Biochem. Biophys. Res. Commun. 33, 551 - 557. 14. Zamir, A,, Miskin, R. & Elson, D. (1969) FEBS Lett. 3,85-88. 15. Monro, R. E., Cerna, J . & Marcker, K. A. (1968) Biochrmistry, 61,1042- 1049. 16. Leder, P. & Burytyn, L. (1966) Biochem. Biophys. Res. Commun. 25,233-238. 17. Monro, R . E. (1971) Methods Enzymol. 20C, 472-480. 18. Celma, M. L., Monro, R. E. & Vazquez, D. (1970) FEBS Lett. 6,273- 211. 19. Vogel, Z., Vogel, T., Zamir, A. & Elson, D. (1971) J . Mol. Biol. 60, 339- 346. 20. Harris, R. & Pestka, S. (1973) J . Biol. Chem. 248, 1168- 1174. 21 Celma, M. L., Monro, R. E. & Vazquez, D. (1971) FEBS Lett. 13,241-251. 22. Staehelin, T., Maglott, D. & Monro, K. E. (1969) Cold Spring Harbor Symp. Quant. Biol. 34, 39-47. 23 Ray, W. J. (1967) Methods Enzymol. 11,490-497. 24. Westhead, E. W. (1972) Methodr Enzymol. 25B, 401-409. 25. Weil, L. (1965) Arch. Biochem. Biophys. 110. 57-68. 26. Vazquez, D. (1974) FEBS Lelf. 40, S63-S84. 27. Cerna, J., Rychlik, I. & Jonak, J. (1973) Eur. J . Biochem. 34, 551 - 556. 28. Lessard, J. L. & Pestka, S. (1972) J. Biol. Chem. 247, 69096912. 29. Maden, B. E. H. & Monro, R. E. (1968) Eur. J . Biochem. 6 , 309 - 316.

K. K. Wan, N. D. Zahid, and R. M. Baxter, Faculty of Pharmacy, University of Toronto, 19 Russell Street, Toronto, Ontario, Canada M5S 1Al

The photochemical inactivation of peptidyl transferase activity.

Eur. J. Biocheni. 58, 397-402 (1975) The Photochemical Inactivation of Peptidyl Transferase Activity Kwong K. WAN, Nasir D. ZAHID, and Ross M . BAXTE...
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