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Studies on structural proteins of the rat liver ribosomes I. Molecular weights of the proteins of large and small subunits
214
Biochimica et Biophysica Acta, 402 (1975) 214--229 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98370
STUDIES ON STRUCTURAL PROTEINS OF THE RAT LIVER RIBOSOMES I. MOLECULAR WEIGHTS OF THE PROTEINS OF LARGE AND SMALL SUBUNITS
KAZUO TER AO and KIKUO OGATA
Department of Biochemistry, Niigata University School of Medicine, Niigata (Japan) (Received February 24th, 1975)
Summary Structural proteins of active 60-S and 40-S subunits of rat liver ribosomes were analysed by two-dimensional polyacrylamide gel electrophoresis. 35 and 29 spots were shown on two-dimensional gel electrophoresis of proteins from large and small subunits, respectively. It was noted that the migration distances of stained proteins with Amido black 10B remained unchanged in the following sodium dodecyl sulfate-acrylamide gel electrophoresis, although some minor degradation and/or aggregation products were observed in the case of several ribosomal proteins, especially of those with high molecular weights. This finding made it possible to measure the molecular weight of each ribosomal protein in the spot on two-dimensional gel electrophoresis by following sodium dodecyl sulfate-acrylamide gel electrophoresis. The molecular weights of the protein components of two liver ribosomal subunits were determined by this 'threedimensional' polyacrylamide gel electrophoresis. The molecular weights of proteins of 40-S subunits ranged from 10 000 to 38 000 and the number average molecular weight was 23 000. The molecular weights of proteins of 60-S subunits ranged from 10 000 to 60 000 and the number average molecular weight was 23 900.
Introduction Our previous experiments in which proteins of the small subunits from EDTA-treated rat liver ribosomes were fractionated by carboxymethylcellulose column chromatography followed by gel filtration on Sephadex G-200, indicated that there are 28 proteins in the 30 S ribosomal subunit and the sum of the molecular weights of the 28 proteins was about 690 000 [1]. These studies led us to investigate the characterization of proteins of the
215 large subunits of rat liver ribosomes. However, because of the large number of the proteins with similar chemical and physical properties, the separation of proteins by the chromatographies described above were found to be very difficult. On the other hand, two-dimensional polyacrylamide gel electrophoresis developed by Kaltschmidt and Wittmann [2,3] greatly facilitated the analysis of the ribosomal proteins owing to its high resolution capacity and this technique has been used by several authors for the analysis of the protein moiety of eukaryotic ribosomes [4--14]. Accordingly, we applied this method for the analysis of the proteins of the active large and small subunits of rat liver ribosomes. Furthermore, it was found that the mobility of protein on polyacrylamide gel electrophoresis in sodium dodecyl sulfate remained unchanged even after staining proteins with Amido black 10B. Therefore, it was thought to be possible to estimate the molecular weight of the individual protein on two-dimensional gel by following sodium dodecyl sulfate-gel electrophoresis. The present paper describes the result of the determination of the molecular weights of proteins of the ribosomal 60-S and 40-S subunits of rat liver by 'three-dimensional' polyacrylamide gel electrophoresis. Materials and Methods
Isolation o f ribosomal subunits Ribosomal subunits were prepared from rat liver by the procedure originally described by Martin and Wool [15] as described previously [16] except that puromycin was used for the dissociation of ribosomes [17]. In brief, ribosomes of rat liver were prepared from the microsomal fraction by a slightly modified method of Rendi and Hultin [18,19]. Ribosomes were incubated in Medium A' [18] with 0.2 mM puromycin at 37°C for 10 rain, and then in a medium containing 1 M KCI, 10 mM MgCl:, 20 mM 2-mercaptoethanol, and 50 mM Tris • HC1 buffer (pH 7.6) at 37°C for 15 min. The suspension (200--250 As 60 units in 2 ml) was layered onto 15--30% linear sucrose gradient containing Medium II (850 mM KCI, 10 mM MgC12, 10 mM 2-mercaptoethanol and 50 mM Tris • HC1 buffer (pH 7.6), and the ribosomal subunits were separated by centrifugation at 95 000 × g for 5 h in a Spinco SW-27 rotor at 26 ° C. Dimerized 40-S subunits in the 60 S fraction were removed by a second sucrose gradient centrifugation in the medium with low concentrations of Mg2÷ and KC1 as described previously [16]. Extraction o f ribosomal proteins Ribosomal proteins were extracted with acetic acid by a modification of the procedure of Hardy et al. [20]. After isolation of the subunits by sucrose gradient centrifugation, the Mg2÷ concentration in the subunit suspension was made 20 mM by addition of 1 M MgC12, and then an equal volume of cold 99% ethanol was added [21]. The mixture was kept at 0°C for 1 h and the subunits were sedimented by centrifugation at 10 000 X g for 10 min. Precipitated ribosomal subunits were suspended in 100 mM MgC12 (about 10 mg per ml). After the addition of 2 vol. of glacial acetic acid, the mixture was stirred for 24 h at 0°C. Ribosomal proteins were obtained by centrifugation of this mixture at 59 000 X g for 30 min. The yield of extraction was more than 90%.
216
Two-dimensional polyacrylamide gel electrophoresis Two-dimensional gel electrophoresis was carried o u t according to the method of Kaltschmidt and Wittmann [2] except that 8% acrylamide gel was used for the first dimensional electrophoresis and 15% acrylamide gel for the second electrophoresis. Acid-soluble ribosomal proteins precipitated with acetone [9], were dissolved in the sample gel solution without acrylamide (10% sucrose, 8 M urea, 2 mM EDTA and Tris • borate buffer, pH 8.6) [2]. After disulfide reduction with 2-mercaptoethanol as described previously [9], ribosomal proteins were directly applied on the first dimensional gel tube. Electrophoresis and staining of the gel with Amido black 10B were carried o u t under the conditions as described previously [9], except that destaining was performed in 7% acetic acid.
Sodium dodecy I sulfa te-acrylam ide gel elec trophoresis After staining with Amido black 10B, gel discs containing protein spots were removed from the gel slab of the two-dimensional electrophoresis with a stainless steel borer with an internal diameter of 6 mm, and then middle layers of the gel discs were treated as follows. They were placed at 5--10°C in 0.01 M phosphate buffer for 2 h, and then in 0.01 M phosphate buffer containing 1% sodium dodecyl sulfate and 50 mM 2-mercaptoethanol for 12 h. Finally, they were incubated in a 0.01 M phosphate buffer containing 0.1% sodium dodecyl sulfate, 50 mM 2-mercaptoethanol, 5% sucrose and Bromphenol blue as a marker for 30 min at 37 ° C. In some later experiments, these incubation procedures were carried out at 37°C for 1 h successively, and the results obtained by those two kinds of the incubation were the same. The incubated gel discs were placed on the top of the sodium dodecyl sulfate-polyacrylamide gel columns (6 × 80 m m ) a s described previously [1]. Electrophoresis was carried out for 5 - 6 h at 8 mA per tube, using Bromphenol blue as a marker. The gel was then stained with 2.5% Coomassie brilliant blue. The molecular weights of the stained materials were calculated by the method of Weber and Osborn [ 2 2 ] . Results
Patterns of proteins of 60-S and 40-S subunits Throughout the present experiments, the purified 60-S ribosomal subunits free from contamination of the 40-S subunits were used. To avoid aggregation of the protein near the origin of two-dimensional gel, disulfide reduction with 2-mercaptoethanol was usually carried out and 1.5--2 mg of proteins of both subunits and 80-S ribosomes were applied to the gel. As shown in Figs 1A and 1B, 35 and 29 protein spots are identified on two-dimensional polyacrylamide gel electrophoresis of 60-S and 40-S ribosomal proteins, respectively. The patterns are represented schematically in Fig. 2. These patterns are similar to those by other workers [6,7,9,12--14]. The protein spots were numbered according to the system of Kaltschmidt and Wittmann [ 3 ]. Proteins of large and small subunits were marked with the letters L and S, respectively. It must be added that disulfide reduction results in disappearance of the spot L l l in addition to the decrease of aggregated materials
217
near the origin. The faint L23 spot becomes distinct after this treatment, although it is uncertain whether the spot L l l is converted to the spot L23. Almost all of the proteins from 60-S and 40-S subunits migrated toward the cathode in the first dimension except that some 40-S proteins (Sal, Sa2
, O0 |
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F i g . 1 A . Two-dimensional electrophoretograms of liver ribosomal proteins. Proteins from 60-S subunits.
218
0
9. 0
01
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Fig. l B . T w o - d i m e n s i o n a l e l e c t r o p h o r e t o g r a m s of liver r i b o s o m a l p r o t e i n s . P r o t e i n s f r o m 40-S subunits.
and Sa3) ran to the a n o d e . When the total ribosomal proteins were analysed by two-dimensional gel electrophoresis, one distinct protein ( T 1 ) * ran t o w a r d the * T h e m o l e c u l a r w e i g h t o f T1 w a s f o u n d to be 14 0 0 0 b y t h e f o l l o w i n g s o d i u m d o d e c y l sulfate-acryla m i d e gel e l e c t r o p h o r e s i s .
219
Q
•
f Q
i
Fig. 1C. T w o - d i m e n s i o n a l e l e c t r o p h o r e t o g r a m s o f liver r i b o s o m a l p r o t e i n s . P r o t e i n s f r o m 80-S r i b o s o m e s .
anode in the first dimension in addition and Fig. 2C). When 60-S proteins were used, the lapping spot with the L3 spot. Usually two-dimensional electrophoretogram of
to S a l , Sa2 and Sa3 proteins (Fig. 1C L3' spot appeared as a partially overthe L3 ° spot was not observed in the total ribosomal proteins and L3' pro-
220 tein showed the same mobility as L3 proteins on the following sodium dodecyl sulfate-acrylamide gel electrophoresis. Therefore, L3' protein is thought ~o he an artificial product derived from L3 protein during the course of isolation of 60-S subunits and preparation of 60-S proteins. Since L7 and $11 spots were more distinct in the electrophoretogram of total ribosomal proteins than in those of both subunits, they may be partially lost during the preparation of these subunits. *ID
+2D Ori( in
-1D
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UJO OC.~O ('~..
L"r~ ~ L I 5
IS
L24
L26
L31 L32 L33
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-2D
Fig. 2 A . S c h e m a t a o f the t w o - d i m e n s i o n a l e l e c t r o p h o r e t o g r a m s o f liver r i b o s o m a l p r o t e i n s . Proteins f r o m 6 0 - S subunits. +2D + 10
Origin
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-2D Fig. 2B. S c h e m a t a o f the t w o - d i m e n t i o n a l e l e c t r o p h o r e t o g r a m s o f liver r i b o s o m a l p r o t e i n s . Proteins f r o m 40-S subunits.
221
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-2D Fig. 2C. S c h e m a t a of the t w o - d i m e n t i o n a l e l e c t r o p h o r e t o g r a m s of liver r i b o s o m a l p r o t e i n s . P r o t e i n s f r o m 80-S r i b o s o m e s .
Measurement of molecular weights of stained proteins by sodium dodecyl sulfate-acrylamide gel electrophoresis It has been well known that the migration distances of proteins in the sodium dodecyl sulfate-acrylamide gel electrophoresis are correlated to their molecular weights. Such a correlation was generally observed also in the cases of Escherichia coli and rat liver ribosomal proteins by comparing with the molecular weights measured by equilibrium sedimentation [23,24]. When the staining of protein with Amido black 10B has no effect on the mobility of the protein on sodium dodecyl sulfate-acrylamide gel electrophoresis, it can be considered that the combination of two-dimensional acrylamide gel electrophoresis and sodium dodecyl sulfate gel electrophoresis may offer an effective procedure for analysis of complex ribosomal proteins. In order to clarify this point, the following experiments were carried out. Bovine serum albumin (Armour), soybean trypsin inhibitor (a gift from Professor Ikenaka), chymotrypsinogen (Schwarz/Mann) and 60 S ribosomal protein of rat liver which was purified by carboxymethylcellulose column chromatography followed by Sephadex G-200 gel filtration, were subjected to one-dimensional acrylamide gel electrophoresis, according to Reisfield et al. [25]. After staining with Amido black 10B, each band was removed and treated with sodium dodecyl sulfate as described in Materials and Methods. The gel disc was then applied to sodium dodecyl sulfate-acrylamide gel column. As shown in Fig. 3, the original proteins treated according to Weber and Osborn [22] and the proteins stained with dye show similar mobilities with each other in the sodium dodecyl sulfate gel electrophoresis. Therefore, molecular weights of stained proteins on the two-dimensional gel may be estimated by following sodium dodecyl sulfate gel electrophoresis.
222
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Fig. 3. E f f e c t o f s t a i n i n g w i t h A m i d o b l a c k o n t h e m i g r a t i o n d i s t a n c e s o f s o m e p r o t e i n s o n s o d i u m d o d e c y l s u l f a t e - 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 . Urea-gel: disc e l e c t r o p h o r e s i s a c c o r d i n g t o t h e m e t h o d o f R e i s f l e l d et al. [ 2 5 ] . S o d i u m d o d e c y l s u l f a t e gel: 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 a c c o r d i n g t o the method o f W e b e r a n d O s b o r n [ 2 2 ] . 1, r i b o s o m a l p r o t e i n ; 2, b o v i n e s e r u m a l b u m i n ; 3, s o y b e a n t r y p s i n i n h i b i t o r ; 4, c h y m o t r y p s i n o g e n ; A , a f t e r s t a i n i n g w i t h A m i d o b l a c k 1 0 B , p r o t e i n b a n d w a s a p p l i e d t o s o d i u m d o d e c y l s u l f a t e gel; B, o r i g i n a l p r o t e i n s a m p l e t r e a t e d w i t h s o d i u m d o d e c y l s u l f a t e w a s a p p l i e d t o s o d i u m d o d e c y l s u l f a t e gel.
Patterns of sodium dodecyl sulfate gel electrophoresis of the ribosomal proteins and their molecular weights Patterns of sodium dodecyl sulfate-acrylamide gel electrophoresis of the individual protein c o m p o n e n t on two-dimensional gel electrophoresis of large and small subunits, are shown in Fig. 4. L l l protein was obtained from the spot on the electrophoresis of the 60-S proteins w i t h o u t the treatment with 2-mercaptoethanol. A number of ribosomal proteins show only one band or in some cases two distinct bands on sodium dodecyl sulfate gel. Two bands are usually observed in the case of the partially overlapping spots on the two-dimensional gel. In Fig. 4, M is used to mark the main c o m p o n e n t and N to mark the c o m p o n e n t derived from the neighboring spots. It must be mentioned, however, that several spots, L1, L2, L4, L5, L6, $2 and $3 which locate near the origin on two-dimensional gel, show several minor components in addition to the main components on sodium dodecyl sulfate gel. However, it was noted that sodium dodecyl sulfate gel electrophoresis of the unstained spots of these proteins (L1, L2, L4 + L5, L6, S2, $3 and $5) performed as described by Lin and Wool [ 2 6 ] , showed only one main c o m p o n e n t (Fig. 5), in agreement with their result. Therefore, the minor components described above may be degradation and/or aggregation-
223
products caused by staining. It must be mentioned t h a t t h e migration distances of main bands were similar between stained and unstained materials. So we used the main bands on the sodium dodecyl sulfate gel for estimation of the molecular weights. The standard proteins for molecular weight determination were those described previously [1]. In most of our experiments molecular weights of proteins were reproducibly estimated with errors less than 10%. The molecular weights of proteins of rat liver 60-S and 40-S subunits are summarized in Table I. The number average molecular weights for proteins of the 40-S and 60-S subunits are 23 000 and 23 900, respectively.
M
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i I
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........ k~
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....... L S
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224 Discussion Recently, detailed studies on numbering proteins of ribosomal subunits of rat liver by employing similar two-dimensional polyacrylamide gel electrophoresis were reported by Sheraton and Wool [6,27]. Their electrophoretograms are very similar to ours and their protein spots are comparable with ours. The numbers of ribosomal proteins estimated in this work, 29 for 40-S subunits and 35 for 60-S subunits, are less than their numbers, 31 for 40-S and 41 for 60-S subunits. When comparing their electrophoretograms with ours, the following differences were observed. --r.
M
M
M
Jb LlS
L20
L21
L22
t
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L23
L24
L25
L26
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e.
L28 Fig. 4.A.
L2g
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L31
L32
L33
L34
L35
L36
225
The $29, $30, $31 (=L20), L1, L2, L25, L39, L40 and L41 spots in their nomenclature are missing in our electrophoretograms, while our $4, Sal and $20 spots were not observed in their electrophoretogram. Furthermore, T1 protein was shown only in our electrophoretogram of total ribosomes and our $22 spot was resolved into two spots (their $23 and $24) in their electrophoretogram. It must be mentioned that their $22, L1, L2, L40 and L41 spots were observed in the special conditions of electrophoresis and their $31 (=L20) spot was observed in the special conditions of subunit preparation. Concerning $29, ~-
M
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S1
Fig. 4.B.
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,
,
226 (4.B.)
e u
u
lllu J
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s~
s~
s~
s~
sm
sin
s~
:{,
',
s~
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Fig. 4. Patterns of sodium dodecyl sulfate gel electrophoresis of the stained protein spots on two-dimensional gels. (A) Proteins from large subunits; (B) proteins from small subunits.
$30 and L39 proteins in the fast moving basic area, were reported to be seen when the time of electrophoresis was shortened. Therefore, it is possible that the differences may have arisen from the conditions of electrophoresis; they used 18% acrylamide gel in their second electrophoresis while we used 15% gel. Concerning our $4 and $20 spots, it should be noted that the corresponding spots were observed in the electrophoretogram of the small subunits from
%:
i, k ¸ !~
L2
L6
s2
s3
s,
Fig. 5. Patterns o f s o d i u m d o d e c y l sulfate gel electrophoresis o f unstained r i b o s o m a l proteins w i t h high m o l e c u l a r w e i g h t o n t w o - d i m e n s i o n a l gel.
227 TABLE I M O L E C U L A R W E I G H T S O F R I B O S O M A L P R O T E I N S F R O M 40-S A N D 60-S S U B U N I T S
40-S
60-S
S 1 34000 S 2 38000 S 3 35000 S 4 35000 S 5 32000 S 6 26000 S 7 37000 S $ 23000 S 9 25000 S10 28000 S11 24000 $12 21 0 0 0 S13 18000 S14 20000 S15 22000 S16 23000 S17 18000 SlS 21000 2]M i = 6 6 8 0 0 0 Mn
=
23000
S19 $20 $21 $22 $23 $24 $25 $26
17000 17000 20000 21000 21000 21000 11000 14000
Sal Sa2 Sa3
22000 14 0 0 0 10000
L 1 54000 L 2 60000 L 3 39000 L 4 40000 L 5 32000 L 6 30000 L 7 25000 L 8 24000 L 9 19000 L10 22000 (L11 20000) L12 27000 L13 29000 L14 23 0 0 0 L15 28000 L16 28000 L17 23000 L18 26000 ~ Mi = 837000 Mn
=
L19 L20 L21 L22 L23 L24 L25 L26 L27 L2S L29 L30 L31 L32 L33 L34 L35 L36
22000 22000 19000 15000 14000 15000 20000 17000 16000 19000 17000 21000 13000 19000 17000 16000 16000 10000
23900
mammalian cells by other workers [7,13]. The spots in the acidic area including Sal protein were found to be faint and variable in staining intensity among the different subunit preparations. Our $22 spot was rather diffuse, although it was not separated into two spots in our conditions of electrophoresis. These discrepancies should be subjected to future investigations. Although the methods employed here may be a most direct approach for the determination of molecular weights of ribosomal proteins of large and small subunits, there are some assumptions to be examined for the accuracy of the results. (1} Purity of ribosomes and ribosomal subunits: The ribosomes used in this work are of high purity as a result of high KC1 washings, as judged from negligible activity in the incorporation of amino acid into proteins from aminoacyl-tRNA in the absence of cell sap [28]. During the process of dissociation of ribosomes into the subunits, further purification may be expected, since high KC1 washing followed by EDTA treatment releases contaminated cell-sap proteins from the ribosomal subunits as described previously [28]. Therefore, the ribosomal proteins used in the present experiments can be considered almost free from contamination with cell-sap proteins. Contamination of 60-S subunits with 40-S subunits could be removed by the second sucrose gradient centrifugation, as judged by the negligible activity of 60-S subunits in poly(U)-dependent polyphenylalanine synthesis in the absence of 40-S subunits [16]. (2) Accuracy of the molecular weight determination with sodium dodecyl sulfate-acrylamide gel electrophoresis: It has been generally established that the molecular weight determined by sodium dodecyl sulfate-acrylamide electrophoresis coincides with that determined by the equilibrium sedimentation and this was shown in the case of E. coli ribosomal proteins [23]. It must be
228 mentioned, however, that the gel-electrophoretic method was reported to be unsatisfactory in the case of highly charged or small proteins [29], and in the case of liver small subunits two exceptional proteins were reported [24]. Our results showed that the mobilities of proteins on sodium dodecyl sulfate-acrylamide gel electrophoresis remained unchanged after staining of the proteins with Amido black 10B. Although partial degradation occurred in the case of stained ribosomal proteins with large molecular weights, the main components on the sodium dodecyl sulfate gel showed the same migration distances as the unstained proteins. It must be added that the molecular weights of ribosomal proteins of E. coli determined by the present methods are generally in good agreement with those reported by Kaltschmidt et al. [23] (data not shown). After completion of the present work, two data were available for the molecular weights of large and small subunits of ribosomes of rat liver [26] and rabbit reticulocyte [14] which were determined by similar methods to those of the present experiments. The number average molecular weights for proteins of small subunits of rat liver and rabbit reticulocyte were reported to be 25 400 and 22 000, respectively, and those for large subunits of rat liver and rabbit reticulocyte 28 000 and 27 000, respectively. When we compared our results (23 000 for the small subunits and 23 900 for the large subunits) with those cited above, the value for the small subunits is similar to that for reticulocyte ribosomal small subunits but that for the large subunits is somewhat lower than the values of liver and reticulocyte large subunits described above.
Acknowledgement This work was supported by funds from the Ministry of Education. Thanks are given to Dr I.G. Wool, University of Chicago, for sending us the manuscript before publication. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Terao, K. a n d O g a t a , K. ( 1 9 7 2 ) Biochim. Biophys. A c t a 2 8 5 , 4 7 3 - - 4 8 2 K a l t s c h m i d t , E. a n d W i t t m a n n , H.G. ( 1 9 6 9 ) Anal. B i o c h e m . 3 6 , 4 0 1 - - 4 1 2 K a l t s c h m i d t , E. a n d W i t t m a n n , H.G. ( 1 9 7 0 ) Proc. Natl. A c a d . Sci. U.S. 67, 1 2 7 6 - - 1 2 8 2 Martini. O.H.W. a n d G o u l d , H.J. ( 1 9 7 1 ) J. Mol. Biol. 6 2 , 4 0 3 - - 4 0 5 H u y n h - V a n - T a n , D e l a u n a y , J. a n d S c h a p i r a , G. ( 1 9 7 1 ) FEBS Lett. 17, 1 6 3 - - 1 6 7 S h e r t o n , C.C. a n d Wool, I.G. ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 4 4 6 0 - - 4 4 6 7 Welfle, H., S t a h l , J. a n d Bielka, H. ( 1 9 7 2 ) FEBS L e t t . 2 6 , 2 2 3 - - 2 3 2 H u l t i n , T. a n d Sjoqvist, A. ( 1 9 7 2 ) Anal. B i o c h e m . 4 6 , 3 4 2 - - 3 4 6 Tsurugi, K., Morita, T. a n d O g a t a , K. ( 1 9 7 3 ) Eur. J. B i o c h e m . 3 2 , 5 5 5 - - 5 6 2 P r a t t , H. a n d Cox, R.A. ( 1 9 7 3 ) B i o c h i m . B i o p h y s . A c t a 3 1 0 , 1 8 8 - - 2 0 4 H a n n a , N., Bellemare, G. a n d G o d i n , C. ( 1 9 7 3 ) Blochim. B i o p h y s . A c t a 3 3 1 , 1 4 1 - - 1 4 5 Chatterjee0 S.K., Kazemie, M. a n d M a t t h a e i , H. ( 1 9 7 3 ) H o p p e - S e y l e r ' s Z. Physiol. C h e m . 3 5 4 , 4 8 1 - 486 Peeters, B., V a n d u f f e l , L., D e p u y d t , A. a n d R o m b a u t S , W. ( 1 9 7 3 ) FEBS L e t t . 3 6 , 2 1 7 - - 2 2 1 T r a u t , R.T., H o w a r d , G.A. a n d T r a u g h , J . A . ( 1 9 7 3 ) in Metabolic I n t e r c o n v e r s i o n o f E n z y m e s (Fischer, E.H., Krebs, E.G., N e u r a t h , H. a n d S t a d t m a n , E.R., eds), pp. 1 5 5 ~ 1 6 4 , Springer-Verlag Martin, T.E. a n d Wool, I.G. ( 1 9 6 8 ) Proc. Natl. A c a d . Sci. U.S. 6 0 , 5 6 9 - - 5 7 4 T e r a o , K. a n d O g a t a , K. ( 1 9 7 0 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 38, 8 0 - - 8 5 Martin, T.E., R o l l e s t o n , F.S., L o w , R.B. a n d Wool, I.G. ( 1 9 6 9 ) J. Mol. Biol. 4 3 , 1 3 5 - - 1 4 9 T e r a o , K., K a t s u m L H., S u g a n o , H. a n d O g a t a , K. ( 1 9 6 7 ) Biochim. B i o p h y s . A c t a 138, 3 6 9 - - 3 8 1 R e n d i , R. a n d Huitin, T. ( 1 9 6 0 ) Exp. Cell Res. 1 9 , 2 5 3 - - 2 6 6
229 20 21 22 23 24 25 26 27 28 29
Hardy, S.J.S., Kurland, C.G., Voynow, P. and Mora, G. (1969) Biochemistry 8, 2 8 9 7 - - 2 9 0 5 Falvey, A.K. and Staehelin, T. (1970) J. Mol. Biol. 53, 1--19 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4 4 0 6 - - 4 4 1 2 Dzionara, M., Kaltsehmidt, E. and Wittmann, H.G. (1970) Proe. Natl. Aead. Sei. U.S. 67, 1909--1913 Westermann, P. and Bielka, H. (1973) Mol. Gen. Genet. 1 2 6 , 3 4 9 - - 3 5 6 Reisfeld, R.A., Lewis, U.J. and Williams° D.E. (1962) Nature 195, 281--283 Lin, A. and Wool, I.G. (1974) Mol. Gen. Genet. 134, 1--6 Sheraton, C. and Wool, I.G. (1974) J. Biol. Chem. 249, 2 2 5 8 - - 2 2 6 7 Terao, K., Tsurugi, K. and Ogata, K. (1974) J. Bioehem. T o k y o 76, 1113--1122 Williams, J.G. and Gratzer, W.B. (1971) J. Chromatogr. 57, 121--125
Biochimica et Biophysica Acta, 402 (1975) 214--229 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98370
STUDIES ON STRUCTURAL PROTEINS OF THE RAT LIVER RIBOSOMES I. MOLECULAR WEIGHTS OF THE PROTEINS OF LARGE AND SMALL SUBUNITS
KAZUO TER AO and KIKUO OGATA
Department of Biochemistry, Niigata University School of Medicine, Niigata (Japan) (Received February 24th, 1975)
Summary Structural proteins of active 60-S and 40-S subunits of rat liver ribosomes were analysed by two-dimensional polyacrylamide gel electrophoresis. 35 and 29 spots were shown on two-dimensional gel electrophoresis of proteins from large and small subunits, respectively. It was noted that the migration distances of stained proteins with Amido black 10B remained unchanged in the following sodium dodecyl sulfate-acrylamide gel electrophoresis, although some minor degradation and/or aggregation products were observed in the case of several ribosomal proteins, especially of those with high molecular weights. This finding made it possible to measure the molecular weight of each ribosomal protein in the spot on two-dimensional gel electrophoresis by following sodium dodecyl sulfate-acrylamide gel electrophoresis. The molecular weights of the protein components of two liver ribosomal subunits were determined by this 'threedimensional' polyacrylamide gel electrophoresis. The molecular weights of proteins of 40-S subunits ranged from 10 000 to 38 000 and the number average molecular weight was 23 000. The molecular weights of proteins of 60-S subunits ranged from 10 000 to 60 000 and the number average molecular weight was 23 900.
Introduction Our previous experiments in which proteins of the small subunits from EDTA-treated rat liver ribosomes were fractionated by carboxymethylcellulose column chromatography followed by gel filtration on Sephadex G-200, indicated that there are 28 proteins in the 30 S ribosomal subunit and the sum of the molecular weights of the 28 proteins was about 690 000 [1]. These studies led us to investigate the characterization of proteins of the
215 large subunits of rat liver ribosomes. However, because of the large number of the proteins with similar chemical and physical properties, the separation of proteins by the chromatographies described above were found to be very difficult. On the other hand, two-dimensional polyacrylamide gel electrophoresis developed by Kaltschmidt and Wittmann [2,3] greatly facilitated the analysis of the ribosomal proteins owing to its high resolution capacity and this technique has been used by several authors for the analysis of the protein moiety of eukaryotic ribosomes [4--14]. Accordingly, we applied this method for the analysis of the proteins of the active large and small subunits of rat liver ribosomes. Furthermore, it was found that the mobility of protein on polyacrylamide gel electrophoresis in sodium dodecyl sulfate remained unchanged even after staining proteins with Amido black 10B. Therefore, it was thought to be possible to estimate the molecular weight of the individual protein on two-dimensional gel by following sodium dodecyl sulfate-gel electrophoresis. The present paper describes the result of the determination of the molecular weights of proteins of the ribosomal 60-S and 40-S subunits of rat liver by 'three-dimensional' polyacrylamide gel electrophoresis. Materials and Methods
Isolation o f ribosomal subunits Ribosomal subunits were prepared from rat liver by the procedure originally described by Martin and Wool [15] as described previously [16] except that puromycin was used for the dissociation of ribosomes [17]. In brief, ribosomes of rat liver were prepared from the microsomal fraction by a slightly modified method of Rendi and Hultin [18,19]. Ribosomes were incubated in Medium A' [18] with 0.2 mM puromycin at 37°C for 10 rain, and then in a medium containing 1 M KCI, 10 mM MgCl:, 20 mM 2-mercaptoethanol, and 50 mM Tris • HC1 buffer (pH 7.6) at 37°C for 15 min. The suspension (200--250 As 60 units in 2 ml) was layered onto 15--30% linear sucrose gradient containing Medium II (850 mM KCI, 10 mM MgC12, 10 mM 2-mercaptoethanol and 50 mM Tris • HC1 buffer (pH 7.6), and the ribosomal subunits were separated by centrifugation at 95 000 × g for 5 h in a Spinco SW-27 rotor at 26 ° C. Dimerized 40-S subunits in the 60 S fraction were removed by a second sucrose gradient centrifugation in the medium with low concentrations of Mg2÷ and KC1 as described previously [16]. Extraction o f ribosomal proteins Ribosomal proteins were extracted with acetic acid by a modification of the procedure of Hardy et al. [20]. After isolation of the subunits by sucrose gradient centrifugation, the Mg2÷ concentration in the subunit suspension was made 20 mM by addition of 1 M MgC12, and then an equal volume of cold 99% ethanol was added [21]. The mixture was kept at 0°C for 1 h and the subunits were sedimented by centrifugation at 10 000 X g for 10 min. Precipitated ribosomal subunits were suspended in 100 mM MgC12 (about 10 mg per ml). After the addition of 2 vol. of glacial acetic acid, the mixture was stirred for 24 h at 0°C. Ribosomal proteins were obtained by centrifugation of this mixture at 59 000 X g for 30 min. The yield of extraction was more than 90%.
216
Two-dimensional polyacrylamide gel electrophoresis Two-dimensional gel electrophoresis was carried o u t according to the method of Kaltschmidt and Wittmann [2] except that 8% acrylamide gel was used for the first dimensional electrophoresis and 15% acrylamide gel for the second electrophoresis. Acid-soluble ribosomal proteins precipitated with acetone [9], were dissolved in the sample gel solution without acrylamide (10% sucrose, 8 M urea, 2 mM EDTA and Tris • borate buffer, pH 8.6) [2]. After disulfide reduction with 2-mercaptoethanol as described previously [9], ribosomal proteins were directly applied on the first dimensional gel tube. Electrophoresis and staining of the gel with Amido black 10B were carried o u t under the conditions as described previously [9], except that destaining was performed in 7% acetic acid.
Sodium dodecy I sulfa te-acrylam ide gel elec trophoresis After staining with Amido black 10B, gel discs containing protein spots were removed from the gel slab of the two-dimensional electrophoresis with a stainless steel borer with an internal diameter of 6 mm, and then middle layers of the gel discs were treated as follows. They were placed at 5--10°C in 0.01 M phosphate buffer for 2 h, and then in 0.01 M phosphate buffer containing 1% sodium dodecyl sulfate and 50 mM 2-mercaptoethanol for 12 h. Finally, they were incubated in a 0.01 M phosphate buffer containing 0.1% sodium dodecyl sulfate, 50 mM 2-mercaptoethanol, 5% sucrose and Bromphenol blue as a marker for 30 min at 37 ° C. In some later experiments, these incubation procedures were carried out at 37°C for 1 h successively, and the results obtained by those two kinds of the incubation were the same. The incubated gel discs were placed on the top of the sodium dodecyl sulfate-polyacrylamide gel columns (6 × 80 m m ) a s described previously [1]. Electrophoresis was carried out for 5 - 6 h at 8 mA per tube, using Bromphenol blue as a marker. The gel was then stained with 2.5% Coomassie brilliant blue. The molecular weights of the stained materials were calculated by the method of Weber and Osborn [ 2 2 ] . Results
Patterns of proteins of 60-S and 40-S subunits Throughout the present experiments, the purified 60-S ribosomal subunits free from contamination of the 40-S subunits were used. To avoid aggregation of the protein near the origin of two-dimensional gel, disulfide reduction with 2-mercaptoethanol was usually carried out and 1.5--2 mg of proteins of both subunits and 80-S ribosomes were applied to the gel. As shown in Figs 1A and 1B, 35 and 29 protein spots are identified on two-dimensional polyacrylamide gel electrophoresis of 60-S and 40-S ribosomal proteins, respectively. The patterns are represented schematically in Fig. 2. These patterns are similar to those by other workers [6,7,9,12--14]. The protein spots were numbered according to the system of Kaltschmidt and Wittmann [ 3 ]. Proteins of large and small subunits were marked with the letters L and S, respectively. It must be added that disulfide reduction results in disappearance of the spot L l l in addition to the decrease of aggregated materials
217
near the origin. The faint L23 spot becomes distinct after this treatment, although it is uncertain whether the spot L l l is converted to the spot L23. Almost all of the proteins from 60-S and 40-S subunits migrated toward the cathode in the first dimension except that some 40-S proteins (Sal, Sa2
, O0 |
•
b
4
O
Op
f
~t z
d
F i g . 1 A . Two-dimensional electrophoretograms of liver ribosomal proteins. Proteins from 60-S subunits.
218
0
9. 0
01
O o
Fig. l B . T w o - d i m e n s i o n a l e l e c t r o p h o r e t o g r a m s of liver r i b o s o m a l p r o t e i n s . P r o t e i n s f r o m 40-S subunits.
and Sa3) ran to the a n o d e . When the total ribosomal proteins were analysed by two-dimensional gel electrophoresis, one distinct protein ( T 1 ) * ran t o w a r d the * T h e m o l e c u l a r w e i g h t o f T1 w a s f o u n d to be 14 0 0 0 b y t h e f o l l o w i n g s o d i u m d o d e c y l sulfate-acryla m i d e gel e l e c t r o p h o r e s i s .
219
Q
•
f Q
i
Fig. 1C. T w o - d i m e n s i o n a l e l e c t r o p h o r e t o g r a m s o f liver r i b o s o m a l p r o t e i n s . P r o t e i n s f r o m 80-S r i b o s o m e s .
anode in the first dimension in addition and Fig. 2C). When 60-S proteins were used, the lapping spot with the L3 spot. Usually two-dimensional electrophoretogram of
to S a l , Sa2 and Sa3 proteins (Fig. 1C L3' spot appeared as a partially overthe L3 ° spot was not observed in the total ribosomal proteins and L3' pro-
220 tein showed the same mobility as L3 proteins on the following sodium dodecyl sulfate-acrylamide gel electrophoresis. Therefore, L3' protein is thought ~o he an artificial product derived from L3 protein during the course of isolation of 60-S subunits and preparation of 60-S proteins. Since L7 and $11 spots were more distinct in the electrophoretogram of total ribosomal proteins than in those of both subunits, they may be partially lost during the preparation of these subunits. *ID
+2D Ori( in
-1D
L4 OLS
UJO OC.~O ('~..
L"r~ ~ L I 5
IS
L24
L26
L31 L32 L33
L34 L55
-2D
Fig. 2 A . S c h e m a t a o f the t w o - d i m e n s i o n a l e l e c t r o p h o r e t o g r a m s o f liver r i b o s o m a l p r o t e i n s . Proteins f r o m 6 0 - S subunits. +2D + 10
Origin
-1D
~$1
O~ 2
C3ss
OSO2
%,
$130
CC)s7
0
512
% o ''$ $16
$19 0
% Q $2~
%.
Q $26
-2D Fig. 2B. S c h e m a t a o f the t w o - d i m e n t i o n a l e l e c t r o p h o r e t o g r a m s o f liver r i b o s o m a l p r o t e i n s . Proteins f r o m 40-S subunits.
221
÷ID
+2D Ori, in
-1 D
s e~:j..8
s~
$8~
L22o
~L6
LT<~
S~
~
SHS ,~-21
L26~' $2~ L32
S~"
L~t~
S~ 'h
L ~
t.N~
-2D Fig. 2C. S c h e m a t a of the t w o - d i m e n t i o n a l e l e c t r o p h o r e t o g r a m s of liver r i b o s o m a l p r o t e i n s . P r o t e i n s f r o m 80-S r i b o s o m e s .
Measurement of molecular weights of stained proteins by sodium dodecyl sulfate-acrylamide gel electrophoresis It has been well known that the migration distances of proteins in the sodium dodecyl sulfate-acrylamide gel electrophoresis are correlated to their molecular weights. Such a correlation was generally observed also in the cases of Escherichia coli and rat liver ribosomal proteins by comparing with the molecular weights measured by equilibrium sedimentation [23,24]. When the staining of protein with Amido black 10B has no effect on the mobility of the protein on sodium dodecyl sulfate-acrylamide gel electrophoresis, it can be considered that the combination of two-dimensional acrylamide gel electrophoresis and sodium dodecyl sulfate gel electrophoresis may offer an effective procedure for analysis of complex ribosomal proteins. In order to clarify this point, the following experiments were carried out. Bovine serum albumin (Armour), soybean trypsin inhibitor (a gift from Professor Ikenaka), chymotrypsinogen (Schwarz/Mann) and 60 S ribosomal protein of rat liver which was purified by carboxymethylcellulose column chromatography followed by Sephadex G-200 gel filtration, were subjected to one-dimensional acrylamide gel electrophoresis, according to Reisfield et al. [25]. After staining with Amido black 10B, each band was removed and treated with sodium dodecyl sulfate as described in Materials and Methods. The gel disc was then applied to sodium dodecyl sulfate-acrylamide gel column. As shown in Fig. 3, the original proteins treated according to Weber and Osborn [22] and the proteins stained with dye show similar mobilities with each other in the sodium dodecyl sulfate gel electrophoresis. Therefore, molecular weights of stained proteins on the two-dimensional gel may be estimated by following sodium dodecyl sulfate gel electrophoresis.
222
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urea
I
..l •
®-
•I
®
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"i
B J
®
t
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® A
So.urn doclecyl sultmo gel
Fig. 3. E f f e c t o f s t a i n i n g w i t h A m i d o b l a c k o n t h e m i g r a t i o n d i s t a n c e s o f s o m e p r o t e i n s o n s o d i u m d o d e c y l s u l f a t e - 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 . Urea-gel: disc e l e c t r o p h o r e s i s a c c o r d i n g t o t h e m e t h o d o f R e i s f l e l d et al. [ 2 5 ] . S o d i u m d o d e c y l s u l f a t e gel: 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 a c c o r d i n g t o the method o f W e b e r a n d O s b o r n [ 2 2 ] . 1, r i b o s o m a l p r o t e i n ; 2, b o v i n e s e r u m a l b u m i n ; 3, s o y b e a n t r y p s i n i n h i b i t o r ; 4, c h y m o t r y p s i n o g e n ; A , a f t e r s t a i n i n g w i t h A m i d o b l a c k 1 0 B , p r o t e i n b a n d w a s a p p l i e d t o s o d i u m d o d e c y l s u l f a t e gel; B, o r i g i n a l p r o t e i n s a m p l e t r e a t e d w i t h s o d i u m d o d e c y l s u l f a t e w a s a p p l i e d t o s o d i u m d o d e c y l s u l f a t e gel.
Patterns of sodium dodecyl sulfate gel electrophoresis of the ribosomal proteins and their molecular weights Patterns of sodium dodecyl sulfate-acrylamide gel electrophoresis of the individual protein c o m p o n e n t on two-dimensional gel electrophoresis of large and small subunits, are shown in Fig. 4. L l l protein was obtained from the spot on the electrophoresis of the 60-S proteins w i t h o u t the treatment with 2-mercaptoethanol. A number of ribosomal proteins show only one band or in some cases two distinct bands on sodium dodecyl sulfate gel. Two bands are usually observed in the case of the partially overlapping spots on the two-dimensional gel. In Fig. 4, M is used to mark the main c o m p o n e n t and N to mark the c o m p o n e n t derived from the neighboring spots. It must be mentioned, however, that several spots, L1, L2, L4, L5, L6, $2 and $3 which locate near the origin on two-dimensional gel, show several minor components in addition to the main components on sodium dodecyl sulfate gel. However, it was noted that sodium dodecyl sulfate gel electrophoresis of the unstained spots of these proteins (L1, L2, L4 + L5, L6, S2, $3 and $5) performed as described by Lin and Wool [ 2 6 ] , showed only one main c o m p o n e n t (Fig. 5), in agreement with their result. Therefore, the minor components described above may be degradation and/or aggregation-
223
products caused by staining. It must be mentioned t h a t t h e migration distances of main bands were similar between stained and unstained materials. So we used the main bands on the sodium dodecyl sulfate gel for estimation of the molecular weights. The standard proteins for molecular weight determination were those described previously [1]. In most of our experiments molecular weights of proteins were reproducibly estimated with errors less than 10%. The molecular weights of proteins of rat liver 60-S and 40-S subunits are summarized in Table I. The number average molecular weights for proteins of the 40-S and 60-S subunits are 23 000 and 23 900, respectively.
M
M
i I
N
•
i I
L~
L 2 ...........~
........ k~
"'~-L--4----'~
....... L S
k 7 ......... C r - - ' L T
! L~O Fig. 4.A.
.......... L f t
"
I,t|
1;,~15" " ~ M
L-I
........
i;.WII . . . . . .
L'I'r'---'IL'I~
"~
224 Discussion Recently, detailed studies on numbering proteins of ribosomal subunits of rat liver by employing similar two-dimensional polyacrylamide gel electrophoresis were reported by Sheraton and Wool [6,27]. Their electrophoretograms are very similar to ours and their protein spots are comparable with ours. The numbers of ribosomal proteins estimated in this work, 29 for 40-S subunits and 35 for 60-S subunits, are less than their numbers, 31 for 40-S and 41 for 60-S subunits. When comparing their electrophoretograms with ours, the following differences were observed. --r.
M
M
M
Jb LlS
L20
L21
L22
t
M
L23
L24
L25
L26
J" "" e.
O"
L27
e.
L28 Fig. 4.A.
L2g
L30
L31
L32
L33
L34
L35
L36
225
The $29, $30, $31 (=L20), L1, L2, L25, L39, L40 and L41 spots in their nomenclature are missing in our electrophoretograms, while our $4, Sal and $20 spots were not observed in their electrophoretogram. Furthermore, T1 protein was shown only in our electrophoretogram of total ribosomes and our $22 spot was resolved into two spots (their $23 and $24) in their electrophoretogram. It must be mentioned that their $22, L1, L2, L40 and L41 spots were observed in the special conditions of electrophoresis and their $31 (=L20) spot was observed in the special conditions of subunit preparation. Concerning $29, ~-
M
i!!i
S1
Fig. 4.B.
M
+
qF
M
•
,
,
226 (4.B.)
e u
u
lllu J
sm
s2o
s~
s~
s~
s~
sm
sin
s~
:{,
',
s~
s~
Fig. 4. Patterns of sodium dodecyl sulfate gel electrophoresis of the stained protein spots on two-dimensional gels. (A) Proteins from large subunits; (B) proteins from small subunits.
$30 and L39 proteins in the fast moving basic area, were reported to be seen when the time of electrophoresis was shortened. Therefore, it is possible that the differences may have arisen from the conditions of electrophoresis; they used 18% acrylamide gel in their second electrophoresis while we used 15% gel. Concerning our $4 and $20 spots, it should be noted that the corresponding spots were observed in the electrophoretogram of the small subunits from
%:
i, k ¸ !~
L2
L6
s2
s3
s,
Fig. 5. Patterns o f s o d i u m d o d e c y l sulfate gel electrophoresis o f unstained r i b o s o m a l proteins w i t h high m o l e c u l a r w e i g h t o n t w o - d i m e n s i o n a l gel.
227 TABLE I M O L E C U L A R W E I G H T S O F R I B O S O M A L P R O T E I N S F R O M 40-S A N D 60-S S U B U N I T S
40-S
60-S
S 1 34000 S 2 38000 S 3 35000 S 4 35000 S 5 32000 S 6 26000 S 7 37000 S $ 23000 S 9 25000 S10 28000 S11 24000 $12 21 0 0 0 S13 18000 S14 20000 S15 22000 S16 23000 S17 18000 SlS 21000 2]M i = 6 6 8 0 0 0 Mn
=
23000
S19 $20 $21 $22 $23 $24 $25 $26
17000 17000 20000 21000 21000 21000 11000 14000
Sal Sa2 Sa3
22000 14 0 0 0 10000
L 1 54000 L 2 60000 L 3 39000 L 4 40000 L 5 32000 L 6 30000 L 7 25000 L 8 24000 L 9 19000 L10 22000 (L11 20000) L12 27000 L13 29000 L14 23 0 0 0 L15 28000 L16 28000 L17 23000 L18 26000 ~ Mi = 837000 Mn
=
L19 L20 L21 L22 L23 L24 L25 L26 L27 L2S L29 L30 L31 L32 L33 L34 L35 L36
22000 22000 19000 15000 14000 15000 20000 17000 16000 19000 17000 21000 13000 19000 17000 16000 16000 10000
23900
mammalian cells by other workers [7,13]. The spots in the acidic area including Sal protein were found to be faint and variable in staining intensity among the different subunit preparations. Our $22 spot was rather diffuse, although it was not separated into two spots in our conditions of electrophoresis. These discrepancies should be subjected to future investigations. Although the methods employed here may be a most direct approach for the determination of molecular weights of ribosomal proteins of large and small subunits, there are some assumptions to be examined for the accuracy of the results. (1} Purity of ribosomes and ribosomal subunits: The ribosomes used in this work are of high purity as a result of high KC1 washings, as judged from negligible activity in the incorporation of amino acid into proteins from aminoacyl-tRNA in the absence of cell sap [28]. During the process of dissociation of ribosomes into the subunits, further purification may be expected, since high KC1 washing followed by EDTA treatment releases contaminated cell-sap proteins from the ribosomal subunits as described previously [28]. Therefore, the ribosomal proteins used in the present experiments can be considered almost free from contamination with cell-sap proteins. Contamination of 60-S subunits with 40-S subunits could be removed by the second sucrose gradient centrifugation, as judged by the negligible activity of 60-S subunits in poly(U)-dependent polyphenylalanine synthesis in the absence of 40-S subunits [16]. (2) Accuracy of the molecular weight determination with sodium dodecyl sulfate-acrylamide gel electrophoresis: It has been generally established that the molecular weight determined by sodium dodecyl sulfate-acrylamide electrophoresis coincides with that determined by the equilibrium sedimentation and this was shown in the case of E. coli ribosomal proteins [23]. It must be
228 mentioned, however, that the gel-electrophoretic method was reported to be unsatisfactory in the case of highly charged or small proteins [29], and in the case of liver small subunits two exceptional proteins were reported [24]. Our results showed that the mobilities of proteins on sodium dodecyl sulfate-acrylamide gel electrophoresis remained unchanged after staining of the proteins with Amido black 10B. Although partial degradation occurred in the case of stained ribosomal proteins with large molecular weights, the main components on the sodium dodecyl sulfate gel showed the same migration distances as the unstained proteins. It must be added that the molecular weights of ribosomal proteins of E. coli determined by the present methods are generally in good agreement with those reported by Kaltschmidt et al. [23] (data not shown). After completion of the present work, two data were available for the molecular weights of large and small subunits of ribosomes of rat liver [26] and rabbit reticulocyte [14] which were determined by similar methods to those of the present experiments. The number average molecular weights for proteins of small subunits of rat liver and rabbit reticulocyte were reported to be 25 400 and 22 000, respectively, and those for large subunits of rat liver and rabbit reticulocyte 28 000 and 27 000, respectively. When we compared our results (23 000 for the small subunits and 23 900 for the large subunits) with those cited above, the value for the small subunits is similar to that for reticulocyte ribosomal small subunits but that for the large subunits is somewhat lower than the values of liver and reticulocyte large subunits described above.
Acknowledgement This work was supported by funds from the Ministry of Education. Thanks are given to Dr I.G. Wool, University of Chicago, for sending us the manuscript before publication. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
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229 20 21 22 23 24 25 26 27 28 29
Hardy, S.J.S., Kurland, C.G., Voynow, P. and Mora, G. (1969) Biochemistry 8, 2 8 9 7 - - 2 9 0 5 Falvey, A.K. and Staehelin, T. (1970) J. Mol. Biol. 53, 1--19 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4 4 0 6 - - 4 4 1 2 Dzionara, M., Kaltsehmidt, E. and Wittmann, H.G. (1970) Proe. Natl. Aead. Sei. U.S. 67, 1909--1913 Westermann, P. and Bielka, H. (1973) Mol. Gen. Genet. 1 2 6 , 3 4 9 - - 3 5 6 Reisfeld, R.A., Lewis, U.J. and Williams° D.E. (1962) Nature 195, 281--283 Lin, A. and Wool, I.G. (1974) Mol. Gen. Genet. 134, 1--6 Sheraton, C. and Wool, I.G. (1974) J. Biol. Chem. 249, 2 2 5 8 - - 2 2 6 7 Terao, K., Tsurugi, K. and Ogata, K. (1974) J. Bioehem. T o k y o 76, 1113--1122 Williams, J.G. and Gratzer, W.B. (1971) J. Chromatogr. 57, 121--125