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Effect of γ-irradiation on Escherichia coli ribosomes: Reversible dissociation
PRELIMINARY NOTES
055
BBA 91209 Effect of y-irrodiotion on Escherichia coli ribosomes: Reversible dissociation
As one of a series of studies on the biological consequences of various treatments which alter physical properties of ribosomes 1-a we describe in this report the effect of y-irradiation on Escherichia coli ribosomes. As far as we know, there has been no work published concerning the effects of ionizing radiation on the physical properties of ribosomes. With respect to the effect of v-irradiation on the functional aspects of ribosomes, KU~AN5 has reported that y-irradiation of E. coli ribosomes results in a decrease in their ability to support poly U-directed phenylalanine incorporation in a cell-free system. The present work confirms and extends his observation by providing a physical basis for this inactivation. In addition, the reversibility of the effect of y-irradiation is described. Ribosomes were prepared from exponentially growing E. coli B as described previously 2 and suspended in 5 mM Mg2+-5o mM K C I - I o mM Tris-HC1 buffer (pH 7.4). Ribosomes were irradiated at room temperature with 137Cs v-rays, at a dose rate of 2.5" lO 3 rad/min. Control samples were exposed to room temperature for the same lengths of time without irradiation. After irradiation, samples were cooled in ice and then analyzed by sucrose density-gradient centrifugation (5-20 % sucrose, I h and 45 rain, 35 ooo rev./min, SW39 rotor, Spinco ultracentrifuge Model L or L 4, 2-4°). As shown in Fig. I, irradiation of E. coli ribosomes resulted in dissociation of 7o-S particles into 3o-S and 5o-S components. The degree of dissociation increased approximately exponentially with dose and was inversely related to the concentration of ribosomes. The precise determination of the kinetics was difficult, however, due to the formation of an "intermediate" component (50-7 ° S, arrow in Fig. IB) similar to that formed upon treatment of E. coli ribosomes with p-chloromercuribenzoate 2 or nitrogen mustard 4. The dissociation caused b y y-irradiation also depended upon the time elapsed after irradiation; dissociation continued slowly for about 2 days at o °. This suggests the formation in the medium of some factor which causes dissociation of ribosomes. In fact when non-irradiated ribosomes were mixed with irradiated buffer, dissociation of ribosomes resulted. Such dissociation, however, was revealed only after incubating the sample at least overnight at o °, and did not exceed 30 % with the buffer irradiated to a dose of lO 5 rad, the highest dose employed in the present work. No dissociation of ribosomes was observed when dilution was made with non-irradiated buffer. Thus, the ribosomal dissociation attained by the irradiation of ribosomes in suspension was much more rapid than that seen when ribosomes were mixed with irradiated medium. This indicates that short-lived radicals or reactive (unstable)~ products formed b y their attack on substances in the medium play the major role in causing ribosomal dissociation. In support of this concept, it was found that the addition of radical scavengers (ethanol (5 %) or/5-mercaptoethanol (6 raM)) to the~ medium resulted in complete protection of ribosomes when analyzed immediately after irradiation. Upon analyzing the same samples after storage at o ° for 12 h, th~ protection b y ethanol decreased to 80 °/o, while that by/~-mercaptoethanol remained. IOO %. I t is possible that the drop of protection b y 20 °/o in the case of ethanol ma3 reflect the effect of direct hits of y-irradiation on ribosomes. /~-Mercaptoethanol, ol Biochim. Biophys. Acta, 157 (1968) 655-657
656
PRELIMINARY NOTES
A
~c
IB It
04
i
7~s
II
02 ~
sos
, ~J
I
: •
.
/1
3os
,
i
< o2~,
.
.
.
3os/"~
~
~°s/t
I_LP{, L tL2,V'L
I
2
3
4
0
I
Effluent
2
3
volume
4
0
[
2
3
(ml)
4
0
I
2
3 4 Effluent
0 I 2 3 volume{ml)
! 4
Fig. I. S e d i m e n t a t i o n p a t t e r n s of E. coli r i b o s o m e s irradiated w i t h two doses of y-rays. A. Control. B. 2. 5 • lO 4 rad. C. 7.5" lO4 rad. S e d i m e n t a t i o n f r o m left to right. R i b o s o m e p r e p a r a t i o n , 0. 5 mg/ml. Fig. 2. Reversal of irradiation-induced ribosomal dissociation b y Mg 2+. A n E. coli r i b o s o m e p r e p a r a t i o n (o.5 m g / m l ) w a s irradiated w i t h y - r a y s to 5" lO4 r a d and a p o r t i o n was analyzed i m m e diately (A). T h e r e m a i n i n g sample was dialyzed o v e r n i g h t a g a i n s t b u f f e r containing twice the c o n c e n t r a t i o n (IO raM) of Mg 2+ a n d 6 mM /5-mercaptoethanol and t h e n analyzed (B). If the irradiated s a m p l e was dialyzed o v e r n i g h t a g a i n s t buffer c o n t a i n i n g the same Mg 2+ c o n c e n t r a t i o n as t h e sample, dissociation w a s slightly m o r e a d v a n c e d t h a n s h o w n in A. S e d i m e n t a t i o n f r o m left to right.
the other hand, is known to be, in addition to a radical scavenger, an effective stabilizer of ribosomal structure 3, and thus is able to prevent or reverse dissociation induced b y direct hits. I t has been shown b y a number of workers ~-9 that irradiation of various aqueous solutions, including Tris buffer, results in the production of H202 which affects a variety of biological materials. We found that treatment of ribosomes with H202 at I" 10 -4 g/ml resulted in slow dissociation at 0 °, but below this concentration there was no discernible effect (details to be published12). According to FREY AND POLLARD s, the amount of H202 produced b y T-irradiation under the conditions similar to those used in the present study (oxygenated Tris buffer, 2.5" lO4-1 . lO 5 rad) is approx. I - l O -5 g/ml which is considerably below the level required to cause ribosomal dissociation. Furthermore, it was found that the addition of catalase to suspending medium, either before or after irradiation, offered no protection against ribosomal dissociation. Thus, in the present system H202 does not appear to play a significant role in dissociation of ribosomes. The observed after-effect must, therefore, be attributable to some other factor, the nature of which is presently unknown. Increasing the Mg 2+ concentration in the suspending medium resulted in less dissociation caused b y v-irradiation. Furthermore, the increase of Mg *+ in an irradiated ribosomal preparation (from 5 to io raM), either by direct addition or b y dialysis, resulted in the re-formation of 7o-S ribosomes. A small but significant amount (lO-2O %) of reversal was also achieved b y the addition of fl-mercaptoethanol. With the samples in which up to 60 °/o of the 7o-S ribosomes had been dissociated (up to 5"IO4 tad, 0.5 mg/ml ribosomes), the simultaneous addition of Mg 2+ (final IO raM) and /5-mercaptoethanol (6 raM) resulted in over 80 °/o reversal at o ° (Fig. 2). Further incubation of such a sample at 37 ° for I h led the reversal to virtual completion (see ref. IO). These re-formed 7o-S particles were stable only in the presence of the elevated amount of Mg ~+ (io raM). When tested at IO mM Mg 2+, the re-formed 7o-S particles were as active as non-irradiated ribosomes in phenylalanine incorporation directed b y poly U in a cell-free system 11 (Table I). The restoration of the full activity was possible only to a given dose level (e.g., 7.5" lO4 rad, 5 mg/ml ribosomes), beyond which there was a Biochim. Biophys. z~cta, 157 (1968) 655-657
057
PRELIMINARY NOTES TABLE I ?4C]PHENYLALANINE INCORPORATION BY"
IRRADIATED
RIBOSOMES
A r i b o s o m e p r e p a r a t i o n (5 m g / m l ) w a s i r r a d i a t e d to a dose of 7.5" lO4 tad. Reversal w a s carried o u t as described in t h e legend of Fig. 2. P h e n y l a l a n i n e i n c o r p o r a t i o n w a s carried o u t a t io mM Mg 2+ essentially according to t h e m e t h o d described b y NIRENBERG AND MATTHAE111.
Ribosomes
[1'C] Phenylalanine incorporated after 9o-min incubation (counts/min per o.I ml)
Non-irradiated I r r a d i a t e d a n d t h e n r e v e r s e d b y Mg 2+
23 800 25 70o
decrease in polypeptide synthesis, in spite of the fact that a substantial amount of 7o-S particles were re-formed after the reversal. We also observed that the phenylalanine-incorporating activity of the re-formed ribosomes decreased drastically upon slight reduction of Mg2+ concentration (from IO to 7.5 mM) in the reaction mixture. This contrasted sharply with non-irradiated ribosomes which exhibited little change, either physically or functionally, at these Mg 2+ concentrations. Thus, the decrease in protein-synthesizing ability of ribosomes after irradiation 5 may be ascribed, at least in part, to the labilization of the 7o-S particles by irradiation, or to the inability of the released subunits to re-associate to form an active complex required for protein synthesis. We wish to thank Mrs. CAROL J. TILLEY and Miss VELMA DICK for technical assistance. This work was supported by grants from the National Cancer Institute of Canada and the Medical Research Council of Canada (MA-I953).
The University o/Alberta Cancer Research Un# (McEachern Laboratory) and Department o/ Biochemistry, Edmonton, Alberta (Canada) Physics Department, Cancer Clinic, Edmonton, Alberta (Canada) i 2 3 4 5 6 7 8 9 IO ii I2
T A I K I TAMAOKI FUMIO MIYAZAWA* KAZUNARI NAKAMURA** J O H N W . SCRIMGER
T. TAMAOKI AND F. MIYAZAWA, J. Mol. Biol., 17 (1966) 537. T. TAMAOKI AND F. MIYAZAWA, J. Mol. Biol., 23 (1967) 35F. MIYAZAWA AND T. TAMAOKI, Biochim. Biophys. Acla, 134 (I967) 47 o. F. MIYAZAWA, V. C. DICK AND T. TAMAOKI, Biochim. Biophys. Acta, 155 (1968) 193. Z. KU~AN, Radiation Res., 27 (1966) 229. H. I. ADLER, Radiation Res., 9 (1958) 451. E. C. POLLARD, M. J. EBERT, C. MILLER, K. KOLACZ AND T. F. BARONE, Science, 147 (1965) lO45. H. E. FREY AND E. C. POLLARD, Radiation Res., 28 (1966) 668. J, J. WEiss, Progr. Nucleic Acid Res. Mol. Biol., 3 (1964) i. F. MIYAZAWA AND T. TAMAOKI, J . Mol. Biol., 24 (1967) 485 . M. W. NIRENBERG AND J. H. MATTHAEI, Proc. Natl. Acad. Sci. U.S., 47 (1961) 1588. K. NAKAMURA AND T. TAMAOKI, Bioehim. Biophys. Acta, in the press. * P r e s e n t address: The N a t i o n a l I n s t i t u t e for Hygienic Sciences, Tokyo, J a p a n . ** P r e s e n t address: The N a t i o n a l I n s t i t u t e for L e p r o s y Research, Tokyo, J a p a n .
Received March 5th, 1968 Biochim. Biophys. Acta, 157 (1968) 655-657
055
BBA 91209 Effect of y-irrodiotion on Escherichia coli ribosomes: Reversible dissociation
As one of a series of studies on the biological consequences of various treatments which alter physical properties of ribosomes 1-a we describe in this report the effect of y-irradiation on Escherichia coli ribosomes. As far as we know, there has been no work published concerning the effects of ionizing radiation on the physical properties of ribosomes. With respect to the effect of v-irradiation on the functional aspects of ribosomes, KU~AN5 has reported that y-irradiation of E. coli ribosomes results in a decrease in their ability to support poly U-directed phenylalanine incorporation in a cell-free system. The present work confirms and extends his observation by providing a physical basis for this inactivation. In addition, the reversibility of the effect of y-irradiation is described. Ribosomes were prepared from exponentially growing E. coli B as described previously 2 and suspended in 5 mM Mg2+-5o mM K C I - I o mM Tris-HC1 buffer (pH 7.4). Ribosomes were irradiated at room temperature with 137Cs v-rays, at a dose rate of 2.5" lO 3 rad/min. Control samples were exposed to room temperature for the same lengths of time without irradiation. After irradiation, samples were cooled in ice and then analyzed by sucrose density-gradient centrifugation (5-20 % sucrose, I h and 45 rain, 35 ooo rev./min, SW39 rotor, Spinco ultracentrifuge Model L or L 4, 2-4°). As shown in Fig. I, irradiation of E. coli ribosomes resulted in dissociation of 7o-S particles into 3o-S and 5o-S components. The degree of dissociation increased approximately exponentially with dose and was inversely related to the concentration of ribosomes. The precise determination of the kinetics was difficult, however, due to the formation of an "intermediate" component (50-7 ° S, arrow in Fig. IB) similar to that formed upon treatment of E. coli ribosomes with p-chloromercuribenzoate 2 or nitrogen mustard 4. The dissociation caused b y y-irradiation also depended upon the time elapsed after irradiation; dissociation continued slowly for about 2 days at o °. This suggests the formation in the medium of some factor which causes dissociation of ribosomes. In fact when non-irradiated ribosomes were mixed with irradiated buffer, dissociation of ribosomes resulted. Such dissociation, however, was revealed only after incubating the sample at least overnight at o °, and did not exceed 30 % with the buffer irradiated to a dose of lO 5 rad, the highest dose employed in the present work. No dissociation of ribosomes was observed when dilution was made with non-irradiated buffer. Thus, the ribosomal dissociation attained by the irradiation of ribosomes in suspension was much more rapid than that seen when ribosomes were mixed with irradiated medium. This indicates that short-lived radicals or reactive (unstable)~ products formed b y their attack on substances in the medium play the major role in causing ribosomal dissociation. In support of this concept, it was found that the addition of radical scavengers (ethanol (5 %) or/5-mercaptoethanol (6 raM)) to the~ medium resulted in complete protection of ribosomes when analyzed immediately after irradiation. Upon analyzing the same samples after storage at o ° for 12 h, th~ protection b y ethanol decreased to 80 °/o, while that by/~-mercaptoethanol remained. IOO %. I t is possible that the drop of protection b y 20 °/o in the case of ethanol ma3 reflect the effect of direct hits of y-irradiation on ribosomes. /~-Mercaptoethanol, ol Biochim. Biophys. Acta, 157 (1968) 655-657
656
PRELIMINARY NOTES
A
~c
IB It
04
i
7~s
II
02 ~
sos
, ~J
I
: •
.
/1
3os
,
i
< o2~,
.
.
.
3os/"~
~
~°s/t
I_LP{, L tL2,V'L
I
2
3
4
0
I
Effluent
2
3
volume
4
0
[
2
3
(ml)
4
0
I
2
3 4 Effluent
0 I 2 3 volume{ml)
! 4
Fig. I. S e d i m e n t a t i o n p a t t e r n s of E. coli r i b o s o m e s irradiated w i t h two doses of y-rays. A. Control. B. 2. 5 • lO 4 rad. C. 7.5" lO4 rad. S e d i m e n t a t i o n f r o m left to right. R i b o s o m e p r e p a r a t i o n , 0. 5 mg/ml. Fig. 2. Reversal of irradiation-induced ribosomal dissociation b y Mg 2+. A n E. coli r i b o s o m e p r e p a r a t i o n (o.5 m g / m l ) w a s irradiated w i t h y - r a y s to 5" lO4 r a d and a p o r t i o n was analyzed i m m e diately (A). T h e r e m a i n i n g sample was dialyzed o v e r n i g h t a g a i n s t b u f f e r containing twice the c o n c e n t r a t i o n (IO raM) of Mg 2+ a n d 6 mM /5-mercaptoethanol and t h e n analyzed (B). If the irradiated s a m p l e was dialyzed o v e r n i g h t a g a i n s t buffer c o n t a i n i n g the same Mg 2+ c o n c e n t r a t i o n as t h e sample, dissociation w a s slightly m o r e a d v a n c e d t h a n s h o w n in A. S e d i m e n t a t i o n f r o m left to right.
the other hand, is known to be, in addition to a radical scavenger, an effective stabilizer of ribosomal structure 3, and thus is able to prevent or reverse dissociation induced b y direct hits. I t has been shown b y a number of workers ~-9 that irradiation of various aqueous solutions, including Tris buffer, results in the production of H202 which affects a variety of biological materials. We found that treatment of ribosomes with H202 at I" 10 -4 g/ml resulted in slow dissociation at 0 °, but below this concentration there was no discernible effect (details to be published12). According to FREY AND POLLARD s, the amount of H202 produced b y T-irradiation under the conditions similar to those used in the present study (oxygenated Tris buffer, 2.5" lO4-1 . lO 5 rad) is approx. I - l O -5 g/ml which is considerably below the level required to cause ribosomal dissociation. Furthermore, it was found that the addition of catalase to suspending medium, either before or after irradiation, offered no protection against ribosomal dissociation. Thus, in the present system H202 does not appear to play a significant role in dissociation of ribosomes. The observed after-effect must, therefore, be attributable to some other factor, the nature of which is presently unknown. Increasing the Mg 2+ concentration in the suspending medium resulted in less dissociation caused b y v-irradiation. Furthermore, the increase of Mg *+ in an irradiated ribosomal preparation (from 5 to io raM), either by direct addition or b y dialysis, resulted in the re-formation of 7o-S ribosomes. A small but significant amount (lO-2O %) of reversal was also achieved b y the addition of fl-mercaptoethanol. With the samples in which up to 60 °/o of the 7o-S ribosomes had been dissociated (up to 5"IO4 tad, 0.5 mg/ml ribosomes), the simultaneous addition of Mg 2+ (final IO raM) and /5-mercaptoethanol (6 raM) resulted in over 80 °/o reversal at o ° (Fig. 2). Further incubation of such a sample at 37 ° for I h led the reversal to virtual completion (see ref. IO). These re-formed 7o-S particles were stable only in the presence of the elevated amount of Mg ~+ (io raM). When tested at IO mM Mg 2+, the re-formed 7o-S particles were as active as non-irradiated ribosomes in phenylalanine incorporation directed b y poly U in a cell-free system 11 (Table I). The restoration of the full activity was possible only to a given dose level (e.g., 7.5" lO4 rad, 5 mg/ml ribosomes), beyond which there was a Biochim. Biophys. z~cta, 157 (1968) 655-657
057
PRELIMINARY NOTES TABLE I ?4C]PHENYLALANINE INCORPORATION BY"
IRRADIATED
RIBOSOMES
A r i b o s o m e p r e p a r a t i o n (5 m g / m l ) w a s i r r a d i a t e d to a dose of 7.5" lO4 tad. Reversal w a s carried o u t as described in t h e legend of Fig. 2. P h e n y l a l a n i n e i n c o r p o r a t i o n w a s carried o u t a t io mM Mg 2+ essentially according to t h e m e t h o d described b y NIRENBERG AND MATTHAE111.
Ribosomes
[1'C] Phenylalanine incorporated after 9o-min incubation (counts/min per o.I ml)
Non-irradiated I r r a d i a t e d a n d t h e n r e v e r s e d b y Mg 2+
23 800 25 70o
decrease in polypeptide synthesis, in spite of the fact that a substantial amount of 7o-S particles were re-formed after the reversal. We also observed that the phenylalanine-incorporating activity of the re-formed ribosomes decreased drastically upon slight reduction of Mg2+ concentration (from IO to 7.5 mM) in the reaction mixture. This contrasted sharply with non-irradiated ribosomes which exhibited little change, either physically or functionally, at these Mg 2+ concentrations. Thus, the decrease in protein-synthesizing ability of ribosomes after irradiation 5 may be ascribed, at least in part, to the labilization of the 7o-S particles by irradiation, or to the inability of the released subunits to re-associate to form an active complex required for protein synthesis. We wish to thank Mrs. CAROL J. TILLEY and Miss VELMA DICK for technical assistance. This work was supported by grants from the National Cancer Institute of Canada and the Medical Research Council of Canada (MA-I953).
The University o/Alberta Cancer Research Un# (McEachern Laboratory) and Department o/ Biochemistry, Edmonton, Alberta (Canada) Physics Department, Cancer Clinic, Edmonton, Alberta (Canada) i 2 3 4 5 6 7 8 9 IO ii I2
T A I K I TAMAOKI FUMIO MIYAZAWA* KAZUNARI NAKAMURA** J O H N W . SCRIMGER
T. TAMAOKI AND F. MIYAZAWA, J. Mol. Biol., 17 (1966) 537. T. TAMAOKI AND F. MIYAZAWA, J. Mol. Biol., 23 (1967) 35F. MIYAZAWA AND T. TAMAOKI, Biochim. Biophys. Acla, 134 (I967) 47 o. F. MIYAZAWA, V. C. DICK AND T. TAMAOKI, Biochim. Biophys. Acta, 155 (1968) 193. Z. KU~AN, Radiation Res., 27 (1966) 229. H. I. ADLER, Radiation Res., 9 (1958) 451. E. C. POLLARD, M. J. EBERT, C. MILLER, K. KOLACZ AND T. F. BARONE, Science, 147 (1965) lO45. H. E. FREY AND E. C. POLLARD, Radiation Res., 28 (1966) 668. J, J. WEiss, Progr. Nucleic Acid Res. Mol. Biol., 3 (1964) i. F. MIYAZAWA AND T. TAMAOKI, J . Mol. Biol., 24 (1967) 485 . M. W. NIRENBERG AND J. H. MATTHAEI, Proc. Natl. Acad. Sci. U.S., 47 (1961) 1588. K. NAKAMURA AND T. TAMAOKI, Bioehim. Biophys. Acta, in the press. * P r e s e n t address: The N a t i o n a l I n s t i t u t e for Hygienic Sciences, Tokyo, J a p a n . ** P r e s e n t address: The N a t i o n a l I n s t i t u t e for L e p r o s y Research, Tokyo, J a p a n .
Received March 5th, 1968 Biochim. Biophys. Acta, 157 (1968) 655-657