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Differences between the protein moieties of active subunits and EDTA-treated subunits of rat liver ribosomes with specific references to A 5 S rRNA · protein complex
230
Biochimica et Biophysica Acta, 402 (1975) 230--237 C) Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98371
DIFFERENCES BETWEEN THE PROTEIN MOIETIES OF ACTIVE SUBUNITS AND EDTA-TREATED SUBUNITS OF RAT LIVER RIBOSOMES WITH SPECIFIC REFERENCES TO A 5 S rRNA • PROTEIN COMPLEX
KAZUO TERAO, YOSHIAKI TAKAHASHI and KIKUO OGATA
Department of Biochemistry, Niigata University School of Medicine, Niigata (Japan) (Received February 24th, 1975)
Summary When active 40 S subunits of rat liver ribosomes were treated with EDTA, the conversion of 40 S subunits to 30 S subunits occurred with partial release of 13 kinds of proteins out of 29 kinds of 40 S proteins. Buy contrast, 60 S subunits completely lost one protein (L3) by EDTA-treatment with concomitant release of the fraction sedimenting at about 7 S (7 S fraction). It was found that the 7 S fraction contained 5 S ribosomal RNA as well as L3 protein having a molecular weight of 38 000. Buoyant density in CsCl of the 7 S fraction was 1.60 g/cm 3 , suggesting that this fraction consisted of RNA and protein at an approximately equal ratio on a weight basis. These findings, taken together with the molecular weights of 5 S rRNA (40 000) and L3 protein, may indicate that the 5 S ribosomal RNA • protein complex from rat liver 60 S subunits consists of one molecule of 5 S ribosomal RNA and one molecule of L3 protein.
Introduction
In the previous report [1], it was shown that the protein content of EDTA-treated 30 S subunits was lower than that of active 40 S subunits while EDTA-treated 50 S subunits and active 60 S subunits were similar in the ratio of protein to RNA. Recently, it was shown that 5 S ribosomal RNA (rRNA) was released from rat liver in large subunits as an RNA • protein complex by EDTA or formaldehyde treatment [2--5]. These observations and the results of the preceding paper [6] concerning the detailed analysis of ribosomal proteins by two-dimensional acrylamide-gel electrophoresis, led us to the identification of released proteins by EDTA-treat-
231 ment of both active subunits of rat liver ribosomes. The work reported here describes the results of the experiments carried o u t for this purpose. Materials and Methods
Preparation of the proteins from active ribosomal subunits and EDTA-treated subunits Active 60 S and 40 S subunits of rat liver ribosomes were isolated as described in our previous reports [6,7]. For the preparation of EDTA-treated subunits, 40 S or 60 S subunits fractionated by sucrose density-gradient centrifugation were dialyzed against a buffer containing 50 mM KC1, 5 mM MgC12 and 20 mM Tris • HC1, pH 7.6, and then concentrated in a collagen bag (Sartorius-Membrane filter GmbH). After addition of one tenth vol. of 250 mM EDTA, the ribosomal subunit suspension was layered on the t o p of a 15--307o sucrose gradient containing 50 mM KC1, 1 mM EDTA and 20 mM Tris • HC1 buffer, pH 7.6, and then centrifuged as described previously [8]. The proteins of the active and the EDTA-treated subunits thus obtained were extracted with 67% acetic acid by a modification of the procedure of Hardy et al. [9] as described previously [6]. Two-dimensional gel electrophoresis was carried out according to the slightly modified method [6] of Kaltschmidt and Wittmann [ 1 0 ] . The gel sizes of the apparatus used in these experiments were 200 X 200 X 4 mm, on a large scale and 150 × 150 X 3 mm, on a small scale. CsC1 b u o y a n t density gradient centrifugation of the ribonucleoprotein particle was carried out according to the m e t h o d of Brunk and Leick [11]. Sodium dodecyl sulfate gel electrophoresis for the proteins was carried o u t according to the method of Weber and Osborn [12]. Polyacrylamide gel electrophoresis of R N A was performed b y the modified m e t h o d of Loening [ 1 3 ] . The acrylamide concentration of the gel for RNA was 570. Results
Two-dimensional gel electrophoretograms o f the proteins from the active ribosomal subunits and the EDTA-treated subunits As shown in Fig. 1, the pattern of two-dimensional acrylamide-gel electrophoresis of proteins of 50 S subunits which were prepared by EDTA-treatment of active 60 S subunits is similar to that of the 60 S proteins, except that one kind of protein, L3, is absent in the EDTA-treated 50 S subunits. The molecular weight of L3 protein was estimated to be 39 000 from sodium dodecyl sulfate gel electrophoresis as described in the preceding paper [6]. On the other hand, the two-dimensional gel electrophoretogram of the proteins from active 40 S subunits and EDTA-treated 30 S subunits showed similar protein spots (Fig. 2(a) and (b)), although the protein content of 30 S subunits was lower than that of 40 S subunits [1,14]. In order to identify proteins released from 40 S subunits by EDTA-treatment, EDTA-treated 40 S subunits were subjected to sucrose density-gradient centrifugation, and the region with a lower S value than 4 S was collected. The
232
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Fig. 1. P a t t e r n of t w o - d i m e n s i o n a l gel e l e c t r o p h o r e s i s of the proteins o f large s u b u n i t s . (a) P r o t e i n s o f 60 S s u b u n i t s . (b) P r o t e i n s of 50 S s u b u n i t s d e r i v e d f r o m 6 0 S s u b u n i t s w i t h E D T A - t r e a t m e n t . The a r r o w s h o w s the d i s a p p e a r a n c e o f L3 p r o t e i n .
proteins extracted from this region by acetic acid were subjected to two-dimensional gel electrophoresis. As shown in Fig. 2(c), 13 protein spots are observed, which correspond to the following 40 S ribosomal proteins described in our preceding papers [6] : $3, $4, $6, $8, S l l , S12, S13, S15, S16, S18, S19, $21 and $25. Characterization o f 5 S r R N A • p r o t e i n c o m p l e x
Since it has been reported that 5 S rRNA is removed from 60 S subunits as ribonucleoprotein particles by EDTA-treatment [2,3,5], the results described above led us to identification of the protein moiety of the 5 S rRNA • protein complex and stoichiometry of the 5 S rRNA • protein complex. When EDTA-treated 60 S subunits were subjected to sucrose density gradient centrifugation, a peak sedimenting at approximately 7 S was detected as shown in Fig. 3. This peak fraction designated as the 7 S fraction was pooled and its RNA and protein moieties were analyzed by polyacrylamide gel electrophoresis. Furthermore, the 7 S fraction was analyzed by buoyant density centrifugation in CsC1. As shown in Fig. 4, the RNA moiety of the 7 S fraction represents only one band at the site corresponding to one component of total ribosomal RNA, which may be 5 S RNA since it migrated slower than isolated tRNA*. For the purpose of analysis of the protein moiety in the 7 S fraction, protein extracted by acetic acid was applied to the sodium dodecyl sulfate * 18 S a n d 28 S r i b o s o m a l R N A s do n o t migrate into the gel under the present e x p e r i m e n t a l conditions.
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Fig. 2. Pattern o f t w o - d i m e n s i o n a l gel e l e c t r o p h o r e s i s o f the proteins o f small subunits. (a) Proteins of 4 0 S s u b u n i t s . (b) Proteins o f 3 0 S subunits derived f r o m 4 0 S subunits w i t h E D T A - t r e a t m e n t . (c) P r o t e i n s released from 4 0 S s u b u n i t s b y E D T A - t r e a t m e n t .
acrylamide gel electrophoresis. As shown in Fig. 5, the pattern of the electrophoresis indicated only one band, which has a molecular weight of 38 000 similar to that of L3 protein described previously [6]. Therefore, it is to be expected that the protein associated with 5 S rRNA is L3 protein. For further identification of the protein, the following experiments were carried out. The 7 S fraction was lyophilized after dialysis against 20 mM ~ • HC1 buffer, pH 7.6 and dissolved in 6 M urea (1 ml). After incubation with pancreatic RNAase (10 pg, Worthington, EC 2.7.7.16) at room
234
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Fig. 3. S e d i m e n t a t i o n p r o f i l e o f 6 0 S s u b u n i t s t r e a t e d w i t h E D T A . E D T A - t r e a t e d 6 0 S s u b u n i t s w e r e p l a c e d o n t h e t o p of a 36 m l l i n e a r s u c r o s e - d e n s i t y g r a d i e n t ( 1 5 - - 3 0 % ) in b u f f e r ( 5 0 m M KCI, 1 m M E D T A a n d 20 m M t r l e t h a n o l a m i n e • HC1, p H 7.6, a n d c e n t r i f u g e d in a S p i n c o SW27 r o t o r at 27 0 0 0 r e v . / m i n f o r 2 0 h. T h e h a t c h e d f r a c t i o n s w e r e p o o l e d for f u r t h e r i n v e s t i g a t i o n s as t h e 7 S f r a c t i o n . T h e a r r o w s h o w s t h e p o s i t i o n o f t R N A as a r e f e r e n c e .
temperature for 2 h, the mixture was dialyzed against 6 M urea and concentrated as described in the legend of Fig. 6. The protein solution was then subjected to two-dimensional acrylamide gel electrophoresis. As shown in Fig. 6, only one spot in the same position as L3 protein appears on the two-dimensional gel electrophoresis. It must be mentioned that this spot is accompanied by a partially overlapping protein spot corresponding to L3' protein, which often appeared on the two-dimensional gel electrophoresis of 60 S proteins b u t n o t of total ribosomal proteins and is assumed to be an artificial product of the protein L3 as discussed in the preceding report [6]. Ribonuclease added to the 7 S fraction showed a very faint spot in a different area of the two
It is well known that when rat liver ribosomes are treated with EDTA, they are dissociated into the large and small subunits and the sedimentation coefficients of both EDTA-treated subunits are lower than those of the active 60 S and 40 S subunits [16,17]. Moreover, a ratio of protein to R N A of the EDTA-treated 30 S subunits is significantly lower than that of the active 40 S subunit, while that of the EDTA-treated 50 S subunits is similar to that of the active 60 S fraction [1,14].
235
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Fig. 4. P a t t e r n o f p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s o f R N A f r o m t h e 7 S f r a c t i o n . (a) T o t a l r i b o s o m a l R N A . (b) P u r i f i e d t R N A f r o m r a t liver. (c) R N A f r o m t h e 7 S f r a c t i o n . Fig. 5. S o d i u m d o d e c y l s u l f a t e gel e l e c t r o p h o r e s i s o f p r o t e i n f r o m t h e 7 S f r a c t i o n . (a) 8 0 S r i b o s o m a l p r o t e i n . (b) P r o t e i n f r o m t h e 7 S f r a c t i o n .
These observations suggest that a larger a m o u n t of proteins is detached from the 40 S subunits by EDTA-treatment than from the 60 S subunits. It was shown that the spots of the released proteins on the two-dimensional gel electrophoresis were 13 out of 29 of 40 S ribosomal proteins. However, since the proteins of the EDTA-treated 30 S subunits retained all protein spots of the 40 S subunits on two-dimensional gel electrophoresis, the partial release of proteins from 40 S subunits by EDTA-treatment seems to occur in a random fashion, in contrast to the case of 60 S subunits. Small subunits of liver ribosomes were shown to be more unstable than large subunits in the presence of
236
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gel e l e c t r o p h o r e s i s o f p r o t e i n f r o m 7 S f r a c t i o n (a) a n d o f t o t a l r i b o s o m a l p r o t e i n s (b). A f t e r R N A a s e t r e a t m e n t , t h e 7 S f r a c t i o n w a s d i a l y z e d a g a i n s t 6 M u r e a a t 0 ° C o v e r n i g h t a n d c o n c e n t r a t e d in a v i s k i n g t u b e t o a b o u t 0.1 m l b y p l a c i n g t h e t u b e in S e p h a d e x G - 2 0 0 p o w d e r . A f t e r t h e p r o t e i n s o l u t i o n w a s d i a l y z e d a g a i n s t t h e s a m p l e s o l u t i o n c o n t a i n i n g 5% s u c r o s e , 8 M u r e a , 2 m M E D T A a n d T r i s - b o r a t e b u f f e r , p H 8 . 6 , it w a s s u b j e c t e d t o t w o - d i m e n s i o n a l a c r y l a m i d e gel e l e c t r o p h o r e s i s s i m u l t a n e o u s l y w i t h t o t a l r i b o s o m a l p r o t e i n s . T h e a r r o w in Fig. 6 ( b ) s h o w s t h e L 3 p r o tein.
EDTA and degraded gradually into heterogeneous materials even in the cold [18]. Accordingly, the released proteins may be located at the surface of the particles with 18 S rRNA which are easily attacked by latent ribonuclease. Thus, these EDTA-treated small subunits may consist of the highly heterogeneous particles as compared with the original 40 S particles. Our previous results [1] that EDTA-treated 30 S subunits retained some activity in poly(U)-
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237
dependent polyphenylalanine synthesis in combination with active 60 S subunits, may be explained by these findings. A 5 S rRNA • protein complex was isolated previously by EDTA-treatment [2,3,5] or formaldehyde-treatment [4] from either rat liver ribosomes or their 60 S subunits, and several workers have reported that a protein associated with 5 S rRNA represented one component in sodium dodecyl sulfate gel electrophoresis. Although a molecular weight of the protein component measured by using the sodium dodecyl sulfate-acrylamide gel electrophoresis is rather variable [2--5], protein isolated by these authors is probably the same component as ours. It is of interest that the 5 S rRNA • protein complex consists of one molecule of 5 S rRNA and one molecule of L3 protein, since recent reports have indicated the importance of the 5 S rRNA • protein complexes in protein biosynthesis [ 5,19].
References 1 Terao, K. and Ogata, K. (1971) Biochim. Biophys. Acta 254, 2 7 8 - - 2 8 5 2 Blobel, G. (1971) Proc. Natl. Acad. Sci. U.S. 68, 1 8 8 1 - - 1 8 8 5 3 Lebleu, B., Marbaix, G., Huez, G., T e m m e r m a n , J., Burny, A. and Chantrenne, H. (1971) Eur. J. Biochem. 19,264---269 4 Petermann, M.L., Hamilton, M.G. and Pavlovec, A. (1972) Biochemistry 11, 2323--2326 5 Gru mmt, F., Gru mmt, I. and Erdmann, V.A. (1974) Eur. J. Biochem. 43, 343--348 6 Terao, K. and Ogata, K. (1975) Biochim. Biophys. Acta 402, 214--229 7 Terao, K. and Ogata0 K. (1970) Biochem. Biophys. Res. Commun. 38, 80--85 8 Terao, K. and Ogata, K. (1972) Bioehim. Biophys. Acta 285, 474--482 9 Hardy, S.J.S., Kurland, C.G., Voynow, P. and Mora, G. (1969) Biochemistry 8, 2897--2905 10 Kaltschmidt, E. and Wittmann, H.G. (1970) Anal. Biochem. 3 6 , 4 0 1 - - 4 1 2 11 Brunk, C.F. and Leick, V. (1969) Biochim. Biophys. Acta 179, 136--144 12 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4 4 0 6 - - 4 4 1 2 13 Loening, U.E. (1967) Biochem. J. 102, 251--257 14 Hamilton, M.G., Pavlovec, A. and Petermann, M.L. (1971) Biochemistry 10, 3424--3427 15 Monier, R. (1972) in The Mechanism of Protein S y n t h e s i s and Its R e g u l a t i o n , (Bosch, L. ed.) pp. 353--394, North-Holland Publishing Co., A m s t e r d a m 16 Peterrnann, M.L. (1964) in The Physical and Chemical Properties of R i b o s o m e s , pp. 1 4 7 - - 1 4 8 , Elsevier Publishing Co., A m s t e r d a m 17 Spirin, A.S. and Gavrilova, L.P. (1969) in The R i b o s o m e , pp. 63--77, Springer-Verlag, Berlin 18 Tashiro, Y. and Siekevitz, P. ( 1 9 6 5 ) J. Mol. Biol. 11, 149--165 19 Nishikawa, K. and Takemura, S. (1974) FEBS Lett. 40, 106--109
Biochimica et Biophysica Acta, 402 (1975) 230--237 C) Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98371
DIFFERENCES BETWEEN THE PROTEIN MOIETIES OF ACTIVE SUBUNITS AND EDTA-TREATED SUBUNITS OF RAT LIVER RIBOSOMES WITH SPECIFIC REFERENCES TO A 5 S rRNA • PROTEIN COMPLEX
KAZUO TERAO, YOSHIAKI TAKAHASHI and KIKUO OGATA
Department of Biochemistry, Niigata University School of Medicine, Niigata (Japan) (Received February 24th, 1975)
Summary When active 40 S subunits of rat liver ribosomes were treated with EDTA, the conversion of 40 S subunits to 30 S subunits occurred with partial release of 13 kinds of proteins out of 29 kinds of 40 S proteins. Buy contrast, 60 S subunits completely lost one protein (L3) by EDTA-treatment with concomitant release of the fraction sedimenting at about 7 S (7 S fraction). It was found that the 7 S fraction contained 5 S ribosomal RNA as well as L3 protein having a molecular weight of 38 000. Buoyant density in CsCl of the 7 S fraction was 1.60 g/cm 3 , suggesting that this fraction consisted of RNA and protein at an approximately equal ratio on a weight basis. These findings, taken together with the molecular weights of 5 S rRNA (40 000) and L3 protein, may indicate that the 5 S ribosomal RNA • protein complex from rat liver 60 S subunits consists of one molecule of 5 S ribosomal RNA and one molecule of L3 protein.
Introduction
In the previous report [1], it was shown that the protein content of EDTA-treated 30 S subunits was lower than that of active 40 S subunits while EDTA-treated 50 S subunits and active 60 S subunits were similar in the ratio of protein to RNA. Recently, it was shown that 5 S ribosomal RNA (rRNA) was released from rat liver in large subunits as an RNA • protein complex by EDTA or formaldehyde treatment [2--5]. These observations and the results of the preceding paper [6] concerning the detailed analysis of ribosomal proteins by two-dimensional acrylamide-gel electrophoresis, led us to the identification of released proteins by EDTA-treat-
231 ment of both active subunits of rat liver ribosomes. The work reported here describes the results of the experiments carried o u t for this purpose. Materials and Methods
Preparation of the proteins from active ribosomal subunits and EDTA-treated subunits Active 60 S and 40 S subunits of rat liver ribosomes were isolated as described in our previous reports [6,7]. For the preparation of EDTA-treated subunits, 40 S or 60 S subunits fractionated by sucrose density-gradient centrifugation were dialyzed against a buffer containing 50 mM KC1, 5 mM MgC12 and 20 mM Tris • HC1, pH 7.6, and then concentrated in a collagen bag (Sartorius-Membrane filter GmbH). After addition of one tenth vol. of 250 mM EDTA, the ribosomal subunit suspension was layered on the t o p of a 15--307o sucrose gradient containing 50 mM KC1, 1 mM EDTA and 20 mM Tris • HC1 buffer, pH 7.6, and then centrifuged as described previously [8]. The proteins of the active and the EDTA-treated subunits thus obtained were extracted with 67% acetic acid by a modification of the procedure of Hardy et al. [9] as described previously [6]. Two-dimensional gel electrophoresis was carried out according to the slightly modified method [6] of Kaltschmidt and Wittmann [ 1 0 ] . The gel sizes of the apparatus used in these experiments were 200 X 200 X 4 mm, on a large scale and 150 × 150 X 3 mm, on a small scale. CsC1 b u o y a n t density gradient centrifugation of the ribonucleoprotein particle was carried out according to the m e t h o d of Brunk and Leick [11]. Sodium dodecyl sulfate gel electrophoresis for the proteins was carried o u t according to the method of Weber and Osborn [12]. Polyacrylamide gel electrophoresis of R N A was performed b y the modified m e t h o d of Loening [ 1 3 ] . The acrylamide concentration of the gel for RNA was 570. Results
Two-dimensional gel electrophoretograms o f the proteins from the active ribosomal subunits and the EDTA-treated subunits As shown in Fig. 1, the pattern of two-dimensional acrylamide-gel electrophoresis of proteins of 50 S subunits which were prepared by EDTA-treatment of active 60 S subunits is similar to that of the 60 S proteins, except that one kind of protein, L3, is absent in the EDTA-treated 50 S subunits. The molecular weight of L3 protein was estimated to be 39 000 from sodium dodecyl sulfate gel electrophoresis as described in the preceding paper [6]. On the other hand, the two-dimensional gel electrophoretogram of the proteins from active 40 S subunits and EDTA-treated 30 S subunits showed similar protein spots (Fig. 2(a) and (b)), although the protein content of 30 S subunits was lower than that of 40 S subunits [1,14]. In order to identify proteins released from 40 S subunits by EDTA-treatment, EDTA-treated 40 S subunits were subjected to sucrose density-gradient centrifugation, and the region with a lower S value than 4 S was collected. The
232
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Fig. 1. P a t t e r n of t w o - d i m e n s i o n a l gel e l e c t r o p h o r e s i s of the proteins o f large s u b u n i t s . (a) P r o t e i n s o f 60 S s u b u n i t s . (b) P r o t e i n s of 50 S s u b u n i t s d e r i v e d f r o m 6 0 S s u b u n i t s w i t h E D T A - t r e a t m e n t . The a r r o w s h o w s the d i s a p p e a r a n c e o f L3 p r o t e i n .
proteins extracted from this region by acetic acid were subjected to two-dimensional gel electrophoresis. As shown in Fig. 2(c), 13 protein spots are observed, which correspond to the following 40 S ribosomal proteins described in our preceding papers [6] : $3, $4, $6, $8, S l l , S12, S13, S15, S16, S18, S19, $21 and $25. Characterization o f 5 S r R N A • p r o t e i n c o m p l e x
Since it has been reported that 5 S rRNA is removed from 60 S subunits as ribonucleoprotein particles by EDTA-treatment [2,3,5], the results described above led us to identification of the protein moiety of the 5 S rRNA • protein complex and stoichiometry of the 5 S rRNA • protein complex. When EDTA-treated 60 S subunits were subjected to sucrose density gradient centrifugation, a peak sedimenting at approximately 7 S was detected as shown in Fig. 3. This peak fraction designated as the 7 S fraction was pooled and its RNA and protein moieties were analyzed by polyacrylamide gel electrophoresis. Furthermore, the 7 S fraction was analyzed by buoyant density centrifugation in CsC1. As shown in Fig. 4, the RNA moiety of the 7 S fraction represents only one band at the site corresponding to one component of total ribosomal RNA, which may be 5 S RNA since it migrated slower than isolated tRNA*. For the purpose of analysis of the protein moiety in the 7 S fraction, protein extracted by acetic acid was applied to the sodium dodecyl sulfate * 18 S a n d 28 S r i b o s o m a l R N A s do n o t migrate into the gel under the present e x p e r i m e n t a l conditions.
233 ((~)
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Fig. 2. Pattern o f t w o - d i m e n s i o n a l gel e l e c t r o p h o r e s i s o f the proteins o f small subunits. (a) Proteins of 4 0 S s u b u n i t s . (b) Proteins o f 3 0 S subunits derived f r o m 4 0 S subunits w i t h E D T A - t r e a t m e n t . (c) P r o t e i n s released from 4 0 S s u b u n i t s b y E D T A - t r e a t m e n t .
acrylamide gel electrophoresis. As shown in Fig. 5, the pattern of the electrophoresis indicated only one band, which has a molecular weight of 38 000 similar to that of L3 protein described previously [6]. Therefore, it is to be expected that the protein associated with 5 S rRNA is L3 protein. For further identification of the protein, the following experiments were carried out. The 7 S fraction was lyophilized after dialysis against 20 mM ~ • HC1 buffer, pH 7.6 and dissolved in 6 M urea (1 ml). After incubation with pancreatic RNAase (10 pg, Worthington, EC 2.7.7.16) at room
234
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Fig. 3. S e d i m e n t a t i o n p r o f i l e o f 6 0 S s u b u n i t s t r e a t e d w i t h E D T A . E D T A - t r e a t e d 6 0 S s u b u n i t s w e r e p l a c e d o n t h e t o p of a 36 m l l i n e a r s u c r o s e - d e n s i t y g r a d i e n t ( 1 5 - - 3 0 % ) in b u f f e r ( 5 0 m M KCI, 1 m M E D T A a n d 20 m M t r l e t h a n o l a m i n e • HC1, p H 7.6, a n d c e n t r i f u g e d in a S p i n c o SW27 r o t o r at 27 0 0 0 r e v . / m i n f o r 2 0 h. T h e h a t c h e d f r a c t i o n s w e r e p o o l e d for f u r t h e r i n v e s t i g a t i o n s as t h e 7 S f r a c t i o n . T h e a r r o w s h o w s t h e p o s i t i o n o f t R N A as a r e f e r e n c e .
temperature for 2 h, the mixture was dialyzed against 6 M urea and concentrated as described in the legend of Fig. 6. The protein solution was then subjected to two-dimensional acrylamide gel electrophoresis. As shown in Fig. 6, only one spot in the same position as L3 protein appears on the two-dimensional gel electrophoresis. It must be mentioned that this spot is accompanied by a partially overlapping protein spot corresponding to L3' protein, which often appeared on the two-dimensional gel electrophoresis of 60 S proteins b u t n o t of total ribosomal proteins and is assumed to be an artificial product of the protein L3 as discussed in the preceding report [6]. Ribonuclease added to the 7 S fraction showed a very faint spot in a different area of the two
It is well known that when rat liver ribosomes are treated with EDTA, they are dissociated into the large and small subunits and the sedimentation coefficients of both EDTA-treated subunits are lower than those of the active 60 S and 40 S subunits [16,17]. Moreover, a ratio of protein to R N A of the EDTA-treated 30 S subunits is significantly lower than that of the active 40 S subunit, while that of the EDTA-treated 50 S subunits is similar to that of the active 60 S fraction [1,14].
235
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These observations suggest that a larger a m o u n t of proteins is detached from the 40 S subunits by EDTA-treatment than from the 60 S subunits. It was shown that the spots of the released proteins on the two-dimensional gel electrophoresis were 13 out of 29 of 40 S ribosomal proteins. However, since the proteins of the EDTA-treated 30 S subunits retained all protein spots of the 40 S subunits on two-dimensional gel electrophoresis, the partial release of proteins from 40 S subunits by EDTA-treatment seems to occur in a random fashion, in contrast to the case of 60 S subunits. Small subunits of liver ribosomes were shown to be more unstable than large subunits in the presence of
236
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gel e l e c t r o p h o r e s i s o f p r o t e i n f r o m 7 S f r a c t i o n (a) a n d o f t o t a l r i b o s o m a l p r o t e i n s (b). A f t e r R N A a s e t r e a t m e n t , t h e 7 S f r a c t i o n w a s d i a l y z e d a g a i n s t 6 M u r e a a t 0 ° C o v e r n i g h t a n d c o n c e n t r a t e d in a v i s k i n g t u b e t o a b o u t 0.1 m l b y p l a c i n g t h e t u b e in S e p h a d e x G - 2 0 0 p o w d e r . A f t e r t h e p r o t e i n s o l u t i o n w a s d i a l y z e d a g a i n s t t h e s a m p l e s o l u t i o n c o n t a i n i n g 5% s u c r o s e , 8 M u r e a , 2 m M E D T A a n d T r i s - b o r a t e b u f f e r , p H 8 . 6 , it w a s s u b j e c t e d t o t w o - d i m e n s i o n a l a c r y l a m i d e gel e l e c t r o p h o r e s i s s i m u l t a n e o u s l y w i t h t o t a l r i b o s o m a l p r o t e i n s . T h e a r r o w in Fig. 6 ( b ) s h o w s t h e L 3 p r o tein.
EDTA and degraded gradually into heterogeneous materials even in the cold [18]. Accordingly, the released proteins may be located at the surface of the particles with 18 S rRNA which are easily attacked by latent ribonuclease. Thus, these EDTA-treated small subunits may consist of the highly heterogeneous particles as compared with the original 40 S particles. Our previous results [1] that EDTA-treated 30 S subunits retained some activity in poly(U)-
1.7
A
.
15E
v.,
< ,
~6
, "~'~ ,
2o
F'rection numbe~
~ 3o
Fig. 7. CsCI b u o y a n t d e n s i t y g r a d i e n t c e n t r i f u g a t i o n o f t h e 7 S f r a c t i o n .
237
dependent polyphenylalanine synthesis in combination with active 60 S subunits, may be explained by these findings. A 5 S rRNA • protein complex was isolated previously by EDTA-treatment [2,3,5] or formaldehyde-treatment [4] from either rat liver ribosomes or their 60 S subunits, and several workers have reported that a protein associated with 5 S rRNA represented one component in sodium dodecyl sulfate gel electrophoresis. Although a molecular weight of the protein component measured by using the sodium dodecyl sulfate-acrylamide gel electrophoresis is rather variable [2--5], protein isolated by these authors is probably the same component as ours. It is of interest that the 5 S rRNA • protein complex consists of one molecule of 5 S rRNA and one molecule of L3 protein, since recent reports have indicated the importance of the 5 S rRNA • protein complexes in protein biosynthesis [ 5,19].
References 1 Terao, K. and Ogata, K. (1971) Biochim. Biophys. Acta 254, 2 7 8 - - 2 8 5 2 Blobel, G. (1971) Proc. Natl. Acad. Sci. U.S. 68, 1 8 8 1 - - 1 8 8 5 3 Lebleu, B., Marbaix, G., Huez, G., T e m m e r m a n , J., Burny, A. and Chantrenne, H. (1971) Eur. J. Biochem. 19,264---269 4 Petermann, M.L., Hamilton, M.G. and Pavlovec, A. (1972) Biochemistry 11, 2323--2326 5 Gru mmt, F., Gru mmt, I. and Erdmann, V.A. (1974) Eur. J. Biochem. 43, 343--348 6 Terao, K. and Ogata, K. (1975) Biochim. Biophys. Acta 402, 214--229 7 Terao, K. and Ogata0 K. (1970) Biochem. Biophys. Res. Commun. 38, 80--85 8 Terao, K. and Ogata, K. (1972) Bioehim. Biophys. Acta 285, 474--482 9 Hardy, S.J.S., Kurland, C.G., Voynow, P. and Mora, G. (1969) Biochemistry 8, 2897--2905 10 Kaltschmidt, E. and Wittmann, H.G. (1970) Anal. Biochem. 3 6 , 4 0 1 - - 4 1 2 11 Brunk, C.F. and Leick, V. (1969) Biochim. Biophys. Acta 179, 136--144 12 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4 4 0 6 - - 4 4 1 2 13 Loening, U.E. (1967) Biochem. J. 102, 251--257 14 Hamilton, M.G., Pavlovec, A. and Petermann, M.L. (1971) Biochemistry 10, 3424--3427 15 Monier, R. (1972) in The Mechanism of Protein S y n t h e s i s and Its R e g u l a t i o n , (Bosch, L. ed.) pp. 353--394, North-Holland Publishing Co., A m s t e r d a m 16 Peterrnann, M.L. (1964) in The Physical and Chemical Properties of R i b o s o m e s , pp. 1 4 7 - - 1 4 8 , Elsevier Publishing Co., A m s t e r d a m 17 Spirin, A.S. and Gavrilova, L.P. (1969) in The R i b o s o m e , pp. 63--77, Springer-Verlag, Berlin 18 Tashiro, Y. and Siekevitz, P. ( 1 9 6 5 ) J. Mol. Biol. 11, 149--165 19 Nishikawa, K. and Takemura, S. (1974) FEBS Lett. 40, 106--109