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Subunits of rat liver ribosomes
J. Mol. Biol. (1964) 9, 610-612
Subunits of Rat Liver Ribosomes Ribosomes from bacteria (Tissieres, Watson, Schlessinger & Hollingworth, 1959), yeast (Chao, 1957), pea seedlings (Tso & Vinograd, 1958), and mammalian reticulocytes (Tso & Vinograd, 1961; Lamfrom & Glowacki, 1962) have been shown to be 70 to 80 s particles, each of which upon removal of magnesium can be dissociated into a large and a small subunit with sedimentation coefficients of approximately 50 sand 30 s, respectively. Purified calf liver ribosomal RNA consists of two species, with sedimentation coefficients of 28 sand 18 s (Hall & Doty, 1959). In bacteria the corresponding material sediments at 23 sand 16 s. Kurland (1960) has shown that the 30 s ribonucleoprotein subunit of Escherichiacoli contains only 16 s RNA, and the 50 s subunit contains all the 23 s RNA as well as some 16 s RNA. It is the purpose of this communication to report that rat liver ribosomes can be separated into two subunits, the smaller containing only 18 s RNA and the larger, 28 s. 2·000 1-000 :i.. E
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Effluent (ml.) FIG. 1. Livers from 150 to 250 g male Charles River "CD" rats were homogenized in a Tefl.onglass Potter-Elvehjem homogenizer (operated mechanically) in 3 vol. of medium containing KCl 0·05 M, magnesium acetate 0·0005 M and tris-HCI 0·001 M, pH 7·8. The supernatant suspension obtained after centrifugation at 15,000 g for 10 min was treated with 0·5% deoxycholate, and ribosomes were sedimented by centrifugation at 150,000 g for 1 hr. The ribosomal pellet was resuspended with a Pobter-Elvehjem homogenizer (operated manually) in sufficient 0·05 M·KCI to yield a solution of 30 to 40 O.D. unitsjml. at 260 mu, The solution was made 0·02 M with respect to sodium pyrophosphate by the addition of 1/9th vol. of a 0·2 M solution, pH 8·0. After 3 min at O°C, Lrnl. samples were layered on 5 to 20% linear sucrose gradients made up in 0·05 M.KCl. Centrifugation was at 63,000 g for 6 hr at 1 to 3°C in the Spinco SW25 head. The gradients were analyzed using a continuous flow-through cuvette and recording spectrophotometer (Gilford M·2000). The figures on the abscissa represent ml. of effluent from the gradient tubes, the smaller numbers representing material from the bottom of the tube, the larger numbers that from the top. (a) Preparation of 50 sand 30 s subunits obtained following treatment with pyrophosphate. (b) Control preparation of 80 s ribosomes (not treated with pyrophosphate). 610
LETTERS TO THE EDITOR
611
Rat liver ribosomes have been found to dissociate into subunits of various sizes, including 46 sand 30 s, under diverse conditions (Hamilton & Petermann, 1959), but the relationship between them is not clear (Petermann & Hamilton, 1961). Using sodium pyrophosphate to complex Mg2 + (Sachs, 1958), we have reproducibly separated rat liver 80 s ribosomes into two discrete subunits with sedimentation coefficients of approximately 50 sand 30 s (Fig. 1). Tashiro, Yphantis, Sabatini & Siekevitz (1964), employing EDTAt as complexing agent, have obtained two similar liver ribosomal subunits with sedimentation coefficients of 47 sand 32 s. Treatment of the subunits with SDS followed by centrifugation through a sucrose gradient containing SDS (Gilbert, 1963) permitted the demonstration that the 30 s particle contains only 18 s RNA and the 50 s particle primarily or entirely 28 s RNA (Fig. 2). We
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FIG. 2. Two fractions, one containing the 50 s, the other the 30 s subunit, were collected following their separation on sucrose gradients (see Fig. l(a) ), made 1% with respect to SDS, and 0·1 M with NaCI, and were precipitated with 2·5 vol. of ethanol overnight at -20°C. A third fraction containing unfractionated 80 s ribosomes was purified with phenol and SDS (Hiatt, 1962). The precipitates were suspended in medium containing 0·1% SDS, 0·1 M-NaCI and 0·01 M-sodium acetate, pH 5·0. One-ml, samples were layered on 5 to 20% linear sucrose gradients made up in the same medium. Centrifugation was at 40,000 g for 13 hr at about 15°C. Abscissa the same as Fig. 1. (a) Material from the 50 s particle fraction, treated as above; (b) material from the 30 s particle fraction, treated as above; (c) ribosomal RNA purified from unfractionated 80 s ribosomes.
have found that the 50 sand 30 s subunits are obtained with sodium pyrophosphate concentrations from 0·01 M to at least 0·05 M, but that they may be degraded to smaller components when the isolation conditions described are not observed. In summary, removal of magnesium from 80 s rat liver ribosomes leads to the separation of two discrete particles of approximately 50 sand 30 s containing, respectively, the 28 s and the 18 s ribosomal RNA molecules. These findings support the application of a common structural model to ribosomes of bacterial, plant and mammalian origin.
t Abbreviations used: EDTA, ethylenediaminetetraacetic acid; SDS, sodium dodecylsulfate.
612
E. C. HENSHAW
The technical assistance of Mr. E. Payson Little is gratefully acknowledged. This work was supported by U.S. Public Health Service Research Grant CA03151, from the National Cancer Institute, and by grants from the American Cancer Society and the Leukemia Society, Inc. Departments of Medicine Harvard Medical School and Beth Israel Hospital Boston, Massachusetts, U.S.A.
EDGAR
C.
HENSHAW
Received 20 May 1964 REFERENCES Chao, F-C. (1957). Arch. Biochem, Biophys. 70, 426. Gilbert, W. (1963). J. Mol. Biol. 6, 389. Hall, B. D. & Doty, P. (1959). J. Mol. Biol. 1, Ill. Hamilton, M. G. & Petermann, M. L. (1959). J. Biol. Ohern. 235, 1441. Hiatt, H. (1962). J. Mol. Biol. 5, 217. Kurland, C. G. (1960). J. Mol. Biol. 2, 83. Lamfrom, H. & Glowacki, E. R. (1962). J. Mol. Biol. 5, 97. Petermann, M. L. & Hamilton, M. G. (1961). In Protein Biosynthesis, ed, by R. J. C. Harris, p. 233. New York: Academic Press. Sachs, H. (1958). J. Biol. Ohern. 233,650. Tashiro, Y., Yphantis, D., Sabatini, D. & Siekevitz, P. (1964). Fed. Proc, 23, 219. Tissieres, A., Watson, J. D., Schlessinger, D. & Hollingworth, B. R. (1959). J. Mol. Biol. 1,221. Tao, P. O. P. & Vinograd, J. (1958). Biochim. biophys. Acta, 30, 570. Tso, P. O. P. & Vinograd, J. (1961), Biochim. biophys. Acta, 49,113.
Subunits of Rat Liver Ribosomes Ribosomes from bacteria (Tissieres, Watson, Schlessinger & Hollingworth, 1959), yeast (Chao, 1957), pea seedlings (Tso & Vinograd, 1958), and mammalian reticulocytes (Tso & Vinograd, 1961; Lamfrom & Glowacki, 1962) have been shown to be 70 to 80 s particles, each of which upon removal of magnesium can be dissociated into a large and a small subunit with sedimentation coefficients of approximately 50 sand 30 s, respectively. Purified calf liver ribosomal RNA consists of two species, with sedimentation coefficients of 28 sand 18 s (Hall & Doty, 1959). In bacteria the corresponding material sediments at 23 sand 16 s. Kurland (1960) has shown that the 30 s ribonucleoprotein subunit of Escherichiacoli contains only 16 s RNA, and the 50 s subunit contains all the 23 s RNA as well as some 16 s RNA. It is the purpose of this communication to report that rat liver ribosomes can be separated into two subunits, the smaller containing only 18 s RNA and the larger, 28 s. 2·000 1-000 :i.. E
o
'D
:. 2·000
...,
(b)
';;;
~
1·000
"0 v
'';:; 0-
o
2·000 1·000
o
10
20
30
Effluent (ml.) FIG. 1. Livers from 150 to 250 g male Charles River "CD" rats were homogenized in a Tefl.onglass Potter-Elvehjem homogenizer (operated mechanically) in 3 vol. of medium containing KCl 0·05 M, magnesium acetate 0·0005 M and tris-HCI 0·001 M, pH 7·8. The supernatant suspension obtained after centrifugation at 15,000 g for 10 min was treated with 0·5% deoxycholate, and ribosomes were sedimented by centrifugation at 150,000 g for 1 hr. The ribosomal pellet was resuspended with a Pobter-Elvehjem homogenizer (operated manually) in sufficient 0·05 M·KCI to yield a solution of 30 to 40 O.D. unitsjml. at 260 mu, The solution was made 0·02 M with respect to sodium pyrophosphate by the addition of 1/9th vol. of a 0·2 M solution, pH 8·0. After 3 min at O°C, Lrnl. samples were layered on 5 to 20% linear sucrose gradients made up in 0·05 M.KCl. Centrifugation was at 63,000 g for 6 hr at 1 to 3°C in the Spinco SW25 head. The gradients were analyzed using a continuous flow-through cuvette and recording spectrophotometer (Gilford M·2000). The figures on the abscissa represent ml. of effluent from the gradient tubes, the smaller numbers representing material from the bottom of the tube, the larger numbers that from the top. (a) Preparation of 50 sand 30 s subunits obtained following treatment with pyrophosphate. (b) Control preparation of 80 s ribosomes (not treated with pyrophosphate). 610
LETTERS TO THE EDITOR
611
Rat liver ribosomes have been found to dissociate into subunits of various sizes, including 46 sand 30 s, under diverse conditions (Hamilton & Petermann, 1959), but the relationship between them is not clear (Petermann & Hamilton, 1961). Using sodium pyrophosphate to complex Mg2 + (Sachs, 1958), we have reproducibly separated rat liver 80 s ribosomes into two discrete subunits with sedimentation coefficients of approximately 50 sand 30 s (Fig. 1). Tashiro, Yphantis, Sabatini & Siekevitz (1964), employing EDTAt as complexing agent, have obtained two similar liver ribosomal subunits with sedimentation coefficients of 47 sand 32 s. Treatment of the subunits with SDS followed by centrifugation through a sucrose gradient containing SDS (Gilbert, 1963) permitted the demonstration that the 30 s particle contains only 18 s RNA and the 50 s particle primarily or entirely 28 s RNA (Fig. 2). We
2·000
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1·000 ::i. E 0
-o
N
~
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(b)
'"
-0
.:5a. 2·000 0
1·000
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FIG. 2. Two fractions, one containing the 50 s, the other the 30 s subunit, were collected following their separation on sucrose gradients (see Fig. l(a) ), made 1% with respect to SDS, and 0·1 M with NaCI, and were precipitated with 2·5 vol. of ethanol overnight at -20°C. A third fraction containing unfractionated 80 s ribosomes was purified with phenol and SDS (Hiatt, 1962). The precipitates were suspended in medium containing 0·1% SDS, 0·1 M-NaCI and 0·01 M-sodium acetate, pH 5·0. One-ml, samples were layered on 5 to 20% linear sucrose gradients made up in the same medium. Centrifugation was at 40,000 g for 13 hr at about 15°C. Abscissa the same as Fig. 1. (a) Material from the 50 s particle fraction, treated as above; (b) material from the 30 s particle fraction, treated as above; (c) ribosomal RNA purified from unfractionated 80 s ribosomes.
have found that the 50 sand 30 s subunits are obtained with sodium pyrophosphate concentrations from 0·01 M to at least 0·05 M, but that they may be degraded to smaller components when the isolation conditions described are not observed. In summary, removal of magnesium from 80 s rat liver ribosomes leads to the separation of two discrete particles of approximately 50 sand 30 s containing, respectively, the 28 s and the 18 s ribosomal RNA molecules. These findings support the application of a common structural model to ribosomes of bacterial, plant and mammalian origin.
t Abbreviations used: EDTA, ethylenediaminetetraacetic acid; SDS, sodium dodecylsulfate.
612
E. C. HENSHAW
The technical assistance of Mr. E. Payson Little is gratefully acknowledged. This work was supported by U.S. Public Health Service Research Grant CA03151, from the National Cancer Institute, and by grants from the American Cancer Society and the Leukemia Society, Inc. Departments of Medicine Harvard Medical School and Beth Israel Hospital Boston, Massachusetts, U.S.A.
EDGAR
C.
HENSHAW
Received 20 May 1964 REFERENCES Chao, F-C. (1957). Arch. Biochem, Biophys. 70, 426. Gilbert, W. (1963). J. Mol. Biol. 6, 389. Hall, B. D. & Doty, P. (1959). J. Mol. Biol. 1, Ill. Hamilton, M. G. & Petermann, M. L. (1959). J. Biol. Ohern. 235, 1441. Hiatt, H. (1962). J. Mol. Biol. 5, 217. Kurland, C. G. (1960). J. Mol. Biol. 2, 83. Lamfrom, H. & Glowacki, E. R. (1962). J. Mol. Biol. 5, 97. Petermann, M. L. & Hamilton, M. G. (1961). In Protein Biosynthesis, ed, by R. J. C. Harris, p. 233. New York: Academic Press. Sachs, H. (1958). J. Biol. Ohern. 233,650. Tashiro, Y., Yphantis, D., Sabatini, D. & Siekevitz, P. (1964). Fed. Proc, 23, 219. Tissieres, A., Watson, J. D., Schlessinger, D. & Hollingworth, B. R. (1959). J. Mol. Biol. 1,221. Tao, P. O. P. & Vinograd, J. (1958). Biochim. biophys. Acta, 30, 570. Tso, P. O. P. & Vinograd, J. (1961), Biochim. biophys. Acta, 49,113.