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A reversible change in the ability of Escherichia coli ribosomes to bind to erythromycin
J. Mol. BioE. (1970) 48, 511-515
LETTERS
TO THE EDITOR
A Reversible Change in the Ability of Escherichiu coli Ribosomes to Bind to Erythromycin The binding of erythromycin to Escherichia co&i ribosomes is apparently dependent upon the incubation temperature and upon the concentration of K+ or NH4+ in the medium. To study the role of these monovalent cations, E. co% ribosomes were pre-incubated at different temperatures in a medium rich or deficient in these cations and the effect of this pre-incubation on the binding ability of the ribosomes was examined. The results suggest that E. co&i ribosomes have a conformation favorable for the binding of an erythromycin molecule at sufficient concentrations of K + or NH, + , but that the conformation becomes unfavorable for such binding with a decrease in the concentration of K + or NH4 + , and that the rate of this reversible conversion depends upon the incubation
temperature. Previous studies in this laboratory demonstrated that erythromycin binds to Escherichia coli ribosomes and that this binding induces a characteristic alteration in. an ability of the ribosomes for poly A-dependent polylysine synthesis (Tanaka 8r Teraoka, 1966,1968). The binding was confirmed by isolation of the complex of radioactive erythromycin with ribosomes from E. coli (Tanaka, Teraoka, Nagira &. Tamaki, 1966a,b), from BaciE1u.s subtilis (Taubman, Jones, Young & Corcoran, 1966) and from Staphylococcus aureus (Mao, 1967). Taubman et al. (1966), using B. subtilis ribosomes, demonstrated that the antibiotic binds to 50 s ribosomal subunits, and this was confirmed for the ribosomes from S. aureus (Mao, 1967) and from E. coli (Tanaka, Teraoka, Tamaki, Otaka & Osawa, 1968). Oleinick & Corcoran (1969) indicated the essential requirement of monovalent cations, such as K + or NH, + , for the binding of erythromycin to B. subtilis ribosomes, a requirement likewise observed for E. coli ribosomes (Tanaka, Teraoka, Tamaki, Otaka & Osawa, Proc. 6th Int. Congr. Chemotherapy, to be published) and for S. aureus ribosomes (Mao & Putterman, 1969). This paper deals with the role of these monovalent cations in the binding and the results suggest their involvement in a reversible conversion of the ribosome conforma.. tion from a form active for the binding of erythromycin to an inactive one. Ribosomes were obtained from E. coli Q13 cells as described in a previous paper (Tanaka & Teraoka, 1968). [14C]Erythromycin was prepared by short-term incubation of washed mycelium of Streptomyces erythreus with [l-14C]propionate according to Kaneda, Butte, Taubman & Corcoran (1962). The specific activity of the [14C]erythromycin preparation was estimated to be 2700 cts/min/l75 ppmoles of erythromycin A by the method of diluting with non-radioactive erythromycin A as described previously (Tanaka et al., 1966b). This value agreed very well with that obtained from microbiological assay. In the routine assay of the binding of erythromycin to ribosomes, 125 ~1. of the reaction mixture, containing ribosomes (3.0 to 2.8 Azeo units as indicated in the Figure legends), 2700 cts/min of [14C]erythromycin, 50 mM-Tris-HCl (pH 7*8), 16 511
612
H.
TERAOKA
mM-magnesium acetate and the indicated amounts of monovalent cation, was incubated at 37°C for 15 minutes unless otherwise specified. After incubationthe reaction tube was immediately immersed in an ice bath and the reaction mixture diluted with 3 ml. of cold buffer containing O-025 M-Tris-HCI (pH 7*8), 0.06 ~-Kc1 and 0.02 M-magnesium acetate. This diluted mixture was poured onto a Millipore filter (type HA 0.45 p, pre-washed with the same buffer) and the filter was washed with 3-ml. portions of the cold buffer five times. The [14C]erythromycin-ribosome complex adsorbed on the filter was counted with a Beckman liquid-scintillation counting system.
FIG. 1. Effect of monovalent cations on the binding of erythromycin to ribosomes. The reactions were carried out at varying concentrations of K+, NH4+ or Na+ by the use of 2.8 Azso units of ribosomes. Other conditions were the same as described under routine assay method.
As expected from previous work (Oleinick & Corcoran, 1969 ; Mao & Putterman, 1969) the binding of erythromycin to ribosomes was strongly dependent upon the K+ or NH,+ concentration but not on the Na+ concentration. As shown in Figure 1, the amount of erythromycin bound to ribosomes reached a maximum (approximately equimolar) at approximately 0.05 ~-Kc1 under routine assay conditions. As indicated by the solid line, NH4+ was a little more efficient than K+ . At lower concentrations binding of erythromycin was not observed of K+ or NH4+, where the maximum under the routine assay conditions, an increasing binding of the antibiotic was seen with increasing erythromycin concentration until approximately equimolar binding of antibiotic to ribosomes was achieved. This indicated that a change in K + or NH, + concentration induces a change in the affinity but not the binding capacity of ribosomes for this antibiot,ic. To obtain further information on this point,, the effect of pre-incubation of E. coli ribosomes at high NH,+ concentration was examined. Figure 2 shows that without pre-incubation, maximum binding of erythromycin to the ribosomes was observed after approximately five minutes of incubation at 37”C, while only a small amount of the antibiotic was bound at 0°C. On the other hand, ribosomes pre-incubated at 37°C in a high concentration of NH, + bound nearly the maximum amount of erythromycin even at O”C, almost immediately after the addition of the antibiotic. The ribosomes pre-incubated at such a high concentration of NH,+ at O”C, however, showed little
LETTERS
TO
THE
EDITOR
513
5 Time (mid
FIG. 2. Time-course of erythromyoin binding to ribosomes with or without pm-incubation. Pre-incubations of ribosomes (3.0 A *so units) were carried out for 15 min at 37 or 0°C in buffer containing 0.24 M-NH,Cl with other components of the usual reaction mixture and then cooled in an ice beth for 5 min. By addition of 2 ~1. of [14C]erythromycin to these pre-incubated preparations, the binding reactions were carried out at 0°C for varying periods of time. Without pre-incubation, the reaction were carried out with 0.07 M-KC1 at 37 or 0°C for varying periods of time by the use of 3.0 A,,,, units of ribosomes. Other conditions were the same as described under routine assay method. -O--O-, Binding at 37°C to non-pre-incubated ribosomes; --e---a--, binding at 0°C to non-pre-incubated ribosomes; -- A --- A --, binding at 0°C to ribosomes pre-incubated at 37°C; --n---A-, binding at 0°C to ribosomes pm-incubated at 0°C.
binding ability under the same condition. These results suggest that some conformational change of ribosomes occurs during pre-incubation, and that in order to acquire the conformation favorable for erythromycin binding, ribosomes must be incubated at high temperature and in a sufficient concentration of K+ or NH,+. The maximum binding of the antibiotic to pre-incubated ribosomes, which was observed at O”C, suggests that the binding reaction of erythromycin itself proceeds very rapidly even at low temperature, but that the conformational change of ribosomes is a relatively slow and temperature-dependent reaction. As shown in Table 1, complete loss of binding ability was seen in ribosomes pre-incubated at 37°C and at an extremely low concentration of K+ (expt. l(a)). The binding activity of the ribosomes was remarkably recovered by a second preincubation at high NH,+ concentration at 37°C (expt. l(c)) though not at 0°C (expt. l(b)). From these results it was assumed that the presence of a large amount of K+ or NH,+ may not always be needed for the binding of erythromycin but that thesr monovalent cations may have an important role in the regulation of the conformational changes of ribosomes. As shown in Table 1 (expt. 2), even in NH,+-deficient medium, ribosomes pre-incubated with high NH, + concentration could bind as much as 607; of the maximum amount (equimolar to ribosomes) of erythromycin at 0°C. This may be interpreted as an alteration in the conformation of ribosomes by pre-incubation at high NH, + concentration such that ribosomes could bind erythromycin even in the medium deficient in K+ or NH,” at 0°C. From the results obtained in this study it may safely be concluded that the binding
H.
614
TERAOKA
TABLE 1
Effect of pre-incubation on the ability of ribosomee to bind to erythromycin Pre-incubation 1st pre-incubation
conditions 2nd pre-incubation
[rW]erythromycin bound (cts/min) (wmoles)
Expt. 1 37”C, low K+ 37”C, low K+ 37”C, low K+
iit; (cl
O”C, high NH4 + 37”C, high NHI+
Expt. 2
-
37”C, high NH.,
40 108 964
2.6 7.1 62.8
568
36.9
Experiment 1: Ribosomes were pre-incubated in ‘i&-magnesium buffer containing 0.006 M-KC1 at 37°C for 15 min and then cooled in an ice bath. A sample of these pre-incubated ribosomes (2.8 Azso units) was assayed 8t 0°C for 10 min for the binding of erythromyoin 8s described below. (8) The binding reaction w&s immediately carried out at 0°C with 0.07 M-KCl. (b) After the second pre-incub8tion was carried out with 0.24 M-NH&I at O”C, the binding reaction ~8s carried out 8s described for ribosomes with pre-incubation in the legend to Fig. 2. (c) The condition was the s&me 8s expt. (b), except that the second pre-incubation w&s carried out at 37°C. Expel-intent 2: Ribosomes were pre-incubated in Tris-magnesium buffer containing 0.2 M-NH,Cl 8t 37°C for 15 min and then cooled in 8n ice bath. A sample of these pre-incubated ribosomes (2.8 Ass0 units) w&s assayed at 0°C for 10 min in the presence of 0.008 M-NH,CI. Other conditions were the same 8s described under routine assay method.
affinity of E. coli ribosomes for erythromycin depends upon the ribosomal conformation which is reversibly altered with changes in the concentration of K + or NH, + , the rate of the alteration being largely dependent upon temperature. These facts are of particular interest in view of the finding of Traub & Nomura (1969) that the reconstitution of biologically active 30 s particles was achieved only after incubation at high concentration of KC1 and at relatively high temperature (37 to 40°C). Furthermore, Miskin, Zamir & Elson (1968) have reported that the activity of peptidyltransferase, which is located specifically on 50 s subunits and responsible for peptide-bond formation, is altered reversibly depending on the concentration of KC or NH,+. Considered together, it may be suggested that K+ and NH,+ have an important role in the function of ribosomes by regulating their conformation, and that the erythromycin binding ability of E. coli ribosomes may reflect in some degree the conformation of ribosomes, especially that of the 50 s subunits. The author the preparation
thanks Dr K. Tanaka for many stimulating discussions and for help in of this manuscript, and Mr M. Tamaki for excellent technical assistance. HIROSHI
Shionogi Research Laboratory Shionogi & Co., Ltd Fukushima-ku, Osaka, J8pan Received
22 July
1969, and in revised
form
15 December
TERAOKA
1969.
REFERENCES Keneda, T., Butte, J. C., Taubman, S. B. & Corcoran, J. W. (1962). 322. Mao, J. C.-H. (1967). Biochem. Pharmacob. 16, 2441. Mao, J. C.-H. & Putterman, M. (1969). J. Mol. Biol. 44, 347.
J. Biol.
Chem. 237,
LETTERS
TO
THE
EDITOR
5 15
Miskin, R., Zamir, A. & Elson, D. (1968). Biochem. Biophys. Res. Comm. 33, 551. Oleinick, N. L. & Corcoran, J. W. (1969). J. Biol. Chem. 244, 727. Tanaka, K. & Teraoka, H. (1966). Biochim. biophys. Acta, 114, 204. Tanaka, K. & Teraoka, H. (1968). J. Biochem., Tokyo, 64, 635. Tanaka, K., Teraoka, H., Nagira, T. & Tamrtki, M. (1966a). J. Biochem., Tokyo, 59, 632. Tanaka, K. Teraoka, H., Nagira, T. & Tamaki, M. (19666). Biochim. biophys. Acta, 123, 435. Tanaka, K., Teraoka, H., Tamaki, M., Otaka, E. & Osawa. S. (1968). Science, 162, 576. Taubman, S. B., Jones, N. R., Young, F. E. & Corcoran, J. W. (1966). Biochim. biophys. Acta, 123, 438. Traub, P. & Nomura, M. (1969). J. Mol. Biol. 40, 391.
LETTERS
TO THE EDITOR
A Reversible Change in the Ability of Escherichiu coli Ribosomes to Bind to Erythromycin The binding of erythromycin to Escherichia co&i ribosomes is apparently dependent upon the incubation temperature and upon the concentration of K+ or NH4+ in the medium. To study the role of these monovalent cations, E. co% ribosomes were pre-incubated at different temperatures in a medium rich or deficient in these cations and the effect of this pre-incubation on the binding ability of the ribosomes was examined. The results suggest that E. co&i ribosomes have a conformation favorable for the binding of an erythromycin molecule at sufficient concentrations of K + or NH, + , but that the conformation becomes unfavorable for such binding with a decrease in the concentration of K + or NH4 + , and that the rate of this reversible conversion depends upon the incubation
temperature. Previous studies in this laboratory demonstrated that erythromycin binds to Escherichia coli ribosomes and that this binding induces a characteristic alteration in. an ability of the ribosomes for poly A-dependent polylysine synthesis (Tanaka 8r Teraoka, 1966,1968). The binding was confirmed by isolation of the complex of radioactive erythromycin with ribosomes from E. coli (Tanaka, Teraoka, Nagira &. Tamaki, 1966a,b), from BaciE1u.s subtilis (Taubman, Jones, Young & Corcoran, 1966) and from Staphylococcus aureus (Mao, 1967). Taubman et al. (1966), using B. subtilis ribosomes, demonstrated that the antibiotic binds to 50 s ribosomal subunits, and this was confirmed for the ribosomes from S. aureus (Mao, 1967) and from E. coli (Tanaka, Teraoka, Tamaki, Otaka & Osawa, 1968). Oleinick & Corcoran (1969) indicated the essential requirement of monovalent cations, such as K + or NH, + , for the binding of erythromycin to B. subtilis ribosomes, a requirement likewise observed for E. coli ribosomes (Tanaka, Teraoka, Tamaki, Otaka & Osawa, Proc. 6th Int. Congr. Chemotherapy, to be published) and for S. aureus ribosomes (Mao & Putterman, 1969). This paper deals with the role of these monovalent cations in the binding and the results suggest their involvement in a reversible conversion of the ribosome conforma.. tion from a form active for the binding of erythromycin to an inactive one. Ribosomes were obtained from E. coli Q13 cells as described in a previous paper (Tanaka & Teraoka, 1968). [14C]Erythromycin was prepared by short-term incubation of washed mycelium of Streptomyces erythreus with [l-14C]propionate according to Kaneda, Butte, Taubman & Corcoran (1962). The specific activity of the [14C]erythromycin preparation was estimated to be 2700 cts/min/l75 ppmoles of erythromycin A by the method of diluting with non-radioactive erythromycin A as described previously (Tanaka et al., 1966b). This value agreed very well with that obtained from microbiological assay. In the routine assay of the binding of erythromycin to ribosomes, 125 ~1. of the reaction mixture, containing ribosomes (3.0 to 2.8 Azeo units as indicated in the Figure legends), 2700 cts/min of [14C]erythromycin, 50 mM-Tris-HCl (pH 7*8), 16 511
612
H.
TERAOKA
mM-magnesium acetate and the indicated amounts of monovalent cation, was incubated at 37°C for 15 minutes unless otherwise specified. After incubationthe reaction tube was immediately immersed in an ice bath and the reaction mixture diluted with 3 ml. of cold buffer containing O-025 M-Tris-HCI (pH 7*8), 0.06 ~-Kc1 and 0.02 M-magnesium acetate. This diluted mixture was poured onto a Millipore filter (type HA 0.45 p, pre-washed with the same buffer) and the filter was washed with 3-ml. portions of the cold buffer five times. The [14C]erythromycin-ribosome complex adsorbed on the filter was counted with a Beckman liquid-scintillation counting system.
FIG. 1. Effect of monovalent cations on the binding of erythromycin to ribosomes. The reactions were carried out at varying concentrations of K+, NH4+ or Na+ by the use of 2.8 Azso units of ribosomes. Other conditions were the same as described under routine assay method.
As expected from previous work (Oleinick & Corcoran, 1969 ; Mao & Putterman, 1969) the binding of erythromycin to ribosomes was strongly dependent upon the K+ or NH,+ concentration but not on the Na+ concentration. As shown in Figure 1, the amount of erythromycin bound to ribosomes reached a maximum (approximately equimolar) at approximately 0.05 ~-Kc1 under routine assay conditions. As indicated by the solid line, NH4+ was a little more efficient than K+ . At lower concentrations binding of erythromycin was not observed of K+ or NH4+, where the maximum under the routine assay conditions, an increasing binding of the antibiotic was seen with increasing erythromycin concentration until approximately equimolar binding of antibiotic to ribosomes was achieved. This indicated that a change in K + or NH, + concentration induces a change in the affinity but not the binding capacity of ribosomes for this antibiot,ic. To obtain further information on this point,, the effect of pre-incubation of E. coli ribosomes at high NH,+ concentration was examined. Figure 2 shows that without pre-incubation, maximum binding of erythromycin to the ribosomes was observed after approximately five minutes of incubation at 37”C, while only a small amount of the antibiotic was bound at 0°C. On the other hand, ribosomes pre-incubated at 37°C in a high concentration of NH, + bound nearly the maximum amount of erythromycin even at O”C, almost immediately after the addition of the antibiotic. The ribosomes pre-incubated at such a high concentration of NH,+ at O”C, however, showed little
LETTERS
TO
THE
EDITOR
513
5 Time (mid
FIG. 2. Time-course of erythromyoin binding to ribosomes with or without pm-incubation. Pre-incubations of ribosomes (3.0 A *so units) were carried out for 15 min at 37 or 0°C in buffer containing 0.24 M-NH,Cl with other components of the usual reaction mixture and then cooled in an ice beth for 5 min. By addition of 2 ~1. of [14C]erythromycin to these pre-incubated preparations, the binding reactions were carried out at 0°C for varying periods of time. Without pre-incubation, the reaction were carried out with 0.07 M-KC1 at 37 or 0°C for varying periods of time by the use of 3.0 A,,,, units of ribosomes. Other conditions were the same as described under routine assay method. -O--O-, Binding at 37°C to non-pre-incubated ribosomes; --e---a--, binding at 0°C to non-pre-incubated ribosomes; -- A --- A --, binding at 0°C to ribosomes pre-incubated at 37°C; --n---A-, binding at 0°C to ribosomes pm-incubated at 0°C.
binding ability under the same condition. These results suggest that some conformational change of ribosomes occurs during pre-incubation, and that in order to acquire the conformation favorable for erythromycin binding, ribosomes must be incubated at high temperature and in a sufficient concentration of K+ or NH,+. The maximum binding of the antibiotic to pre-incubated ribosomes, which was observed at O”C, suggests that the binding reaction of erythromycin itself proceeds very rapidly even at low temperature, but that the conformational change of ribosomes is a relatively slow and temperature-dependent reaction. As shown in Table 1, complete loss of binding ability was seen in ribosomes pre-incubated at 37°C and at an extremely low concentration of K+ (expt. l(a)). The binding activity of the ribosomes was remarkably recovered by a second preincubation at high NH,+ concentration at 37°C (expt. l(c)) though not at 0°C (expt. l(b)). From these results it was assumed that the presence of a large amount of K+ or NH,+ may not always be needed for the binding of erythromycin but that thesr monovalent cations may have an important role in the regulation of the conformational changes of ribosomes. As shown in Table 1 (expt. 2), even in NH,+-deficient medium, ribosomes pre-incubated with high NH, + concentration could bind as much as 607; of the maximum amount (equimolar to ribosomes) of erythromycin at 0°C. This may be interpreted as an alteration in the conformation of ribosomes by pre-incubation at high NH, + concentration such that ribosomes could bind erythromycin even in the medium deficient in K+ or NH,” at 0°C. From the results obtained in this study it may safely be concluded that the binding
H.
614
TERAOKA
TABLE 1
Effect of pre-incubation on the ability of ribosomee to bind to erythromycin Pre-incubation 1st pre-incubation
conditions 2nd pre-incubation
[rW]erythromycin bound (cts/min) (wmoles)
Expt. 1 37”C, low K+ 37”C, low K+ 37”C, low K+
iit; (cl
O”C, high NH4 + 37”C, high NHI+
Expt. 2
-
37”C, high NH.,
40 108 964
2.6 7.1 62.8
568
36.9
Experiment 1: Ribosomes were pre-incubated in ‘i&-magnesium buffer containing 0.006 M-KC1 at 37°C for 15 min and then cooled in an ice bath. A sample of these pre-incubated ribosomes (2.8 Azso units) was assayed 8t 0°C for 10 min for the binding of erythromyoin 8s described below. (8) The binding reaction w&s immediately carried out at 0°C with 0.07 M-KCl. (b) After the second pre-incub8tion was carried out with 0.24 M-NH&I at O”C, the binding reaction ~8s carried out 8s described for ribosomes with pre-incubation in the legend to Fig. 2. (c) The condition was the s&me 8s expt. (b), except that the second pre-incubation w&s carried out at 37°C. Expel-intent 2: Ribosomes were pre-incubated in Tris-magnesium buffer containing 0.2 M-NH,Cl 8t 37°C for 15 min and then cooled in 8n ice bath. A sample of these pre-incubated ribosomes (2.8 Ass0 units) w&s assayed at 0°C for 10 min in the presence of 0.008 M-NH,CI. Other conditions were the same 8s described under routine assay method.
affinity of E. coli ribosomes for erythromycin depends upon the ribosomal conformation which is reversibly altered with changes in the concentration of K + or NH, + , the rate of the alteration being largely dependent upon temperature. These facts are of particular interest in view of the finding of Traub & Nomura (1969) that the reconstitution of biologically active 30 s particles was achieved only after incubation at high concentration of KC1 and at relatively high temperature (37 to 40°C). Furthermore, Miskin, Zamir & Elson (1968) have reported that the activity of peptidyltransferase, which is located specifically on 50 s subunits and responsible for peptide-bond formation, is altered reversibly depending on the concentration of KC or NH,+. Considered together, it may be suggested that K+ and NH,+ have an important role in the function of ribosomes by regulating their conformation, and that the erythromycin binding ability of E. coli ribosomes may reflect in some degree the conformation of ribosomes, especially that of the 50 s subunits. The author the preparation
thanks Dr K. Tanaka for many stimulating discussions and for help in of this manuscript, and Mr M. Tamaki for excellent technical assistance. HIROSHI
Shionogi Research Laboratory Shionogi & Co., Ltd Fukushima-ku, Osaka, J8pan Received
22 July
1969, and in revised
form
15 December
TERAOKA
1969.
REFERENCES Keneda, T., Butte, J. C., Taubman, S. B. & Corcoran, J. W. (1962). 322. Mao, J. C.-H. (1967). Biochem. Pharmacob. 16, 2441. Mao, J. C.-H. & Putterman, M. (1969). J. Mol. Biol. 44, 347.
J. Biol.
Chem. 237,
LETTERS
TO
THE
EDITOR
5 15
Miskin, R., Zamir, A. & Elson, D. (1968). Biochem. Biophys. Res. Comm. 33, 551. Oleinick, N. L. & Corcoran, J. W. (1969). J. Biol. Chem. 244, 727. Tanaka, K. & Teraoka, H. (1966). Biochim. biophys. Acta, 114, 204. Tanaka, K. & Teraoka, H. (1968). J. Biochem., Tokyo, 64, 635. Tanaka, K., Teraoka, H., Nagira, T. & Tamrtki, M. (1966a). J. Biochem., Tokyo, 59, 632. Tanaka, K. Teraoka, H., Nagira, T. & Tamaki, M. (19666). Biochim. biophys. Acta, 123, 435. Tanaka, K., Teraoka, H., Tamaki, M., Otaka, E. & Osawa. S. (1968). Science, 162, 576. Taubman, S. B., Jones, N. R., Young, F. E. & Corcoran, J. W. (1966). Biochim. biophys. Acta, 123, 438. Traub, P. & Nomura, M. (1969). J. Mol. Biol. 40, 391.