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Preparative two-dimensional polyacrylamide gel electrophoresis of rat liver ribosomal proteins and determination of their amino acid compositions
418
Biochimica et Biophysica Acta, 519 (1978) 418--427 © Elsevier/North-Holland Biomedical Press
BBA 99188
PREPARATIVE TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS OF RAT LIVER RIBOSOMAL PROTEINS AND DETERMINATION OF THEIR AMINO ACID COMPOSITIONS *
M. GOERL, H. WELFLE and H. BIELKA
Central Institute o f Molecular Biology of the Academy of Sciences of the G.D.R., Department o f Cell Physiology, 1115 Berlin-Buch (Germany) (Received September 26th, 1977) (Revised manuscript received December 20th, 1977)
Summary 1. By enlarging the dimensions of the gels used in the'usual analytical twodimensional polyacrylamide gel electrophoresis it is possible to separate much larger amounts of ribosomal protein in comparison to analytical separations. 15 mg of protein mixture of small or large subunits of rat liver ribosomes can be separated by this procedure. 2. The positions of the proteins in the two~limensional patterns are identified with a special staining procedure. The proteins are eluted from the gels with SDS/phosphate buffers. 3. The purity of the extracted proteins was tested by one-dimensional SDSpolyacrylamide gel electrophoresis and two
Introduction The two
* Paper No. XXVI o f t h e series: Proteins o f animal ribosomes. Abbreviation: SDS, s o d i u m d o d e c y l sulphate.
419 were used in the analytical two
Preparation of ribosomal proteins. Rat liver ribosomes were prepared from the postmitochondrial supernatant of homogenates of rat liver as described earlier [5]. The crude ribosomes were dissociated essentially according to Blobel and Sabatini [6] by incubation with puromycin in the presence of 5 mM Tris/ 500 mM KC1/1 mM MgC12/0.5 mM ZnC12/10 mM ~-mercaptoethanol, pH 7.7. The subunits were separated on a large scale by zonal centrifugation according to Eikenberry et al. [7] using the B15 titanium rotor (Beckman) as already described [5]. The ribosomal proteins were extracted from the subunits with 0.25 M HC1, precipitated with acetone and stored at --20°C [2]. Two-dimensional polyacrylamide gel electrophoresis on preparative scale. Compositions of gels and buffers are the same as in the analytical two
420 buffer (in each case 5 1 100 mM sodium phosphate/0.1% SDS, pH 7.1) was changed after 24, 72 and 96 h. The buffer circulated between the electrode chambers during the run. 2-D System C. In the first dimension we used gel composition and buffers as described by Lastick et al. [9] (4% gels, pH 8.6). In the second dimension the electrophoretic conditions were the same as described for 2-D system A. Localisation of the single proteins in the gel slab. For localisation of the proteins the gels were stained either in a thin slab cut from the gel slab or at the surface of one side of the gel slab with 0.01% Coomassie Brilliant Blue G 250 in 12% trichloroacetic acid. Extraction of the proteins. Gel pieces containing the proteins were crushed and dialysed against different buffers in the following manner: (1) 100 mM sodium phosphate, pH 7.1/0.05% SDS/10 mM ~-mercaptoethanol (3 X 500 ml, 20 h); (2) 10 mM sodium phosphate, pH 7.1/ 0.01% SDS/10 mM ~-mercaptoethanol (3 X 500 ml, 6 h); (3) 1 mM sodium phosphate, pH 7.1/0.005% SDS/10 mM ~-mercaptoethanol (3 X 500 ml, 20 h). After dialysis the solutions were filtered and lyo'phflized. Characterisation of the isolated proteins by one-dimensional SDS electrophoresis. Gels and electrophoresis buffer were of the same composition as the gels and buffers used in the second dimension of the system B. Gels of 8 cm length and 6 mm diameter were used. An aliquot of the lyophilized material (about 5%) was resolved in 40/~l 10 mM sodium phosphate, pH 7.1/8 M urea/l% SDS/ 1% ~-mercaptoethanol at room temperature for 2 h. Bromophenol blue was added to the samples as marker dye and 20 ttl of the protein solution were applied to the gels. Electrophoresis was performed vor 6 h at 45 V. The proteins were stained with Coomassie Brilliant Blue and scanned at 600 nm with the Gilford 2400-2 spectrophotometer. Test of purity of the isolated proteins by two-dimensional electrophoresis. The lyophilized proteins were dissolved in 0.6 ml 0.25 N HC1/10 mM ~-mercaptoethanol in each case and 50 ttg bovine serum albumin were added. The proteins were precipitated with 5% trichloroacetic acid, washed at first with 5% trichloroacetic acid/10 mM ~-mercaptoethanol and then several times with acetone. The proteins were dissolved in 50 ttl 8 M urea/10 mM ~-mercaptoethanol at 20°C overnight. The proteins were reduced by incubation with 5 ttl 10 mM 1,4
421 Results
Separation and purity of the proteins The two
422
2
4
lip
,w
9
8
10
12
qlB
qlD
14
15a
16 q
18
~i
24 / 25 Q
28
29
Fig. 1. A n a l y t i c a l 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 s i s o f i s o l a t e d r i b o s o m a l P r o t e i n s o f t h e s m a l l s u b u n i t . 1 0 - # g p r o t e i n p o r t i o n s w h i c h w e r e t z e a t e d w i t h a c e t o n e t o r e m o v e SDS w e r e a n a l y z e d in t h e 2-D s y s t e m A . ~ n ~ a n e q u i p m e n t f o r a n a l y t i c a l s e p a r a t i o n s a t 1 3 0 V f o r 4 h in t h e first a n d 4 0 V a n d 16 h in t h e s e c o n d d i m e n s i o n . T h e P r o t e i n s w e r e s t a i n e d w i t h C o o m a s a t e Brilliant Blue R 2 5 0 .
423
Amino acid composition of the proteins separated by preparative two-dimensional electrophoresis. As known from the literature [12--14], proteins isolated by polyacrylamide gel electrophoresis are suitable for the determination of their amino acid compositions. In order to check whether also the preparative two~limensional electrophoresis procedure used by us is appropriate to obtain correct values for the amino acid composition of proteins after two~iimensional electrophoresis, we first of all analyzed the amino acid compositions of lysozyme and ribonuclease, extracted from the gels slabs after two~limensional electrophoresis. In Table I, the corresponding values for both enzymes, untreated as well as after twodimensional electrophoresis, are given and compared with values known from the literature [15,16]. With the exception of proline and isoleucine, the values are in fairly good agreement. This demonstrates that proteins prepared by two-dimensional electrophoresis in polyacrylamide gel are well suitable for the determination of their amino acid composition. Probes of polyacrylamide taken from 2-D gel slabs, dialyzed, lyophilized and hydrolyzed under the same conditions as for amino acid analysis did not reveal any ninhydrin positive material in the positions of the amino acids. In Tables II and III the amino acid compositions of single ribosomal proteins prepared by two~limensional electrophoresis are demonstrated. The amino acid compositions of the ribosomal proteins from rat liver are fairly similar, as described already in former papers [18--20]. On the other hand, there are sigTABLE I AMINO ACID COMPOSITION OF UNTREATED AND ELECTROPHORETICALLY ZYME AND RIBONUCLEASE A IN COMPARISON TO THE LITERATURE
PREPARED LYSO-
T h e values are e x p r e s s e d in m o l / 1 0 0 t o o l a m i n o acids, M e t , Cys a n d Trp w e r e n o t d e t e r m i n e d , b u t t a k e n i n t o a c c o u n t for c o r r e c t i o n o f o u r data. T h e value o f L e u is so l o w b e c a u s e o f t h e short h y d r o l y s i s t i m e o f 1 6 h. n.d., n o t d e t e r m i n e d . Lysozyme ref. 1 5
Lys H~ A~ A~ Thr Ser G~ Do Gly Ah Cys Val Met He Leu Tyr Phe ~p
Ribonuclease Untreated
Electrophoretically prepared
ref. 16
8.1 3.2 3.2 12.1
Untreated
8.2 3.0 3.4 12.1
Electrophoretically prepared
4.7 0.8 9.5 16.3
4.9 0.9 8.8 15.3
4.3 0.9 8.0 14.9
7.7 3.1 3.2 12.6
5.4
5.2
5.3
8.1
7.7
8.4
7.8
7.2
8.2
12.1
11.1
12.3
3.9 1.6 9.3 9.3 6.2 4.7 1.6 4.7 6.2 2.3 2.3 4.7
4.3 3.7 9.1 8.9 n.d. 4.2 n.d. 3.7 6.4 2.5 2.6 n.d.
4.5 3.3 9.7 8.5 n.d. 4.2 n.d. 3.7 6.0 2.7 2.5 n.d.
9.7 3.2 2.4 9.7 6.5 7.3 3.2 2.4 1.6 4.8 2.4 0
9.7 5.9 2.8 9.4 n.d. 6.5 n.d. 1.2 1.8 5.1 2.4
9.6 3.6 2.4 9.7 n.d. 6.8 n.& 1.4 2.0 5.1 2.4
--
--
Lys
8.6 8.6 11.8 10.2 12.7 13.4 11.1 11.4 9.3 8.0 12.2 8.3 7.8 9.7 9.8 7.3 9.7 5.8 17.6 9.7 9.5 4.6 6,2 12.5
Protein
$1 2 3 4 7 9 10 11 12 13 14 16 17 18 20 21 22 23 26 27 28 29 30 31
1.8 1.7 2.1 2.8 2.4 2.0 1.9 2.5 2.3 4.0 2.1 1.7 2.4 3.8 2.9 3,1 2.7 2.6 1.5 2.5 4.7 0.7 3.0 2.0
His
5.3 7.3 6.3 9.1 5.6 12.7 7.0 10.4 12.2 9.6 8.2 8.8 8.8 10.8 9.3 7.8 8.0 8.0 5.8 12.8 6.5 14.7 7.9 6.6
Arg
6.8 6.8 10.1 10.4 11.2 7.7 8.1 8.8 8.8 8.4 8.6 10~ 7.9 6.8 8.5 10.2 7.7 14.1 10.9 9.5 6.2 8,2 8.6 7.6
Asp
7.2 5.8 6.1 7.4 7.0 5.2 4.2 4.9 3.4 5.1 7.2 7.0 6.2 4.9 4.8 4.7 9.5 3.0 4.2 2.5 7.6 9.3 3.2 4.9
Thr
T h e values are e x p r e s s e d in t o o l / 1 0 0 m o l a m i n o acids.
6.4 5.7 5.6 4.4 6.0 5.8 7.4 6.5 5.5 5.6 5.3 5.9 8.2 6.3 6.0 8.1 7.9 10.3 7.8 5.6 8.6 7.8 11.6 13.2
Set
8.3 12.1 12.0 7.6 11.6 11.9 13.7 11.7 11.4 10.2 11.0 13.0 11.4 12.1 11.0 11.5 11.1 9.9 7.5 8.1 12.9 14.1 11.9 10.5
Glu
5.4 5.4 3.9 4.9 2.4 4,3 4.3 3.5 3.4 3.3 4.2 4.3 4.4 4.9 3.6 3.1 6.0 1.8 4.6 9.6 6.3 3.1 2.6 4.0
Pro
16.1 11.1 9.5 9.8 10.9 7.2 7.5 11.9 10.4 14.3 9.8 9.2 13.1 9.5 12.2 12.2 8.3 12.4 10.9 7.8 9.5 9.0 17.8 16.6
GIy
9.4 7.6 6.6 6.1 5.9 6.9 6.1 6.1 6.0 7.6 5.4 5.7 9.7 5.3 9.8 6.5 5.8 10.7 9.0 11.5 5.1 3.9 6.6 9.0
Ala
7.0 9.0 8.9 5.7 7.2 6.1 7.9 4.9 6.7 5.7 6.6 7.7 5.5 6.2 8.1 6.6 7.1 5.6 5.5 8.0 4.8 10.5 3.9 5.7
Val
5.1 5.3 4.7 7.8 5.0 4.1 5.9 4.8 4.8 3.8 6.1 6.4 4.4 4.4 2.2 4.7 7.6 5.7 3.0 4.4 2.4 3.4 3.9 0.9
Ile
8.0 8.8 7.9 8.5 7.3 9.5 9.3 8.5 10.8 8.0 6.8 7.5 6.8 9.4 7.9 8,1 6.4 4.0 8.7 4.0 8.4 8.8 6.6 2.8
Leu
AMINO ACID COMPOSITIONS OF THE ISOLATED PROTEINS OF THE SMALL SUBUNIT OF RAT LIVER RIBOSOMES
T A B L E II
1.3 1.5 1.1 1.3 1.0 1.7 0.6 1.9 1.7 2.5 2.0 1.9 1.0 2.1 1.2 1.1 0.4 1.6 1.7 1.7 3.6 0.6 2.5 1.2
Tyr
3.8 3.3 3.7 4.1 3.5 2.0 5.2 2.4 3.3 3.9 4.2 3.0 2.7 3.7 2.9 4.6 2.0 4.4 1.7 2.6 4.1 1.8 3.6 2.6
Phe
2 3 2 2 4 2 5 3 4 2 3 4 2 3 2 1 4 3 2 2 1 2 1 1
Number of estimations
bo
L1 2 3 4 7 8 13 14 16 24 25 26 27 29 30 31 33 34 38
tein
Pro-
10.0 10.0 11.1 12.7 10~ 9.3 7.9 7.4 11.9 10.0 13.9 12.0 9.2 10.0 11.7 13.1 12.6 17.1 13.6
Lys
3.5 2.1 1.8 2.3 3.2 2.9 2.2 2.9 2.9 2.0 3.2 2.2 2.3 2.6 2.1 2.9 1.8 1.5 1.8
His
7.7 9.0 9.2 8.4 9.9 5.8 8.2 5.7 8.0 5.1 10.4 6.0 10.6 7.4 9.2 10.8 12.5 11.1 5.7
Arg
8.4 8.5 11.3 6.3 7.3 9.8 9.2 11.2 7.8 6.2 9.2 8.2 8.6 8.9 10.8 9.0 5.1 6.2 8.4
Asp
6.5 5.6 4.3 5.8 4.7 5.6 4.8 5.4 4.7 4.1 4.1 5.9 6.3 3.3 6.1 4.0 6.0 5.1 4.7
Thr
T h e v a l u e s are e x p r e s s e d in t o o l / 1 0 0 t o o l a m i n o acids.
6.5 6.9 4.0 6.9 5.8 9.5 6.8 7.6 7.0 14.0 8.2 7.9 9.7 8.1 6.1 6.7 8.4 5.8 11.0
Set
10.7 9.6 11.3 9.9 9.3 12.5 13.3 10.7 14.5 13.9 11.6 10.6 8.9 11.2 8.7 9.3 9.7 10.8 10.9
Glu
3.9 5.6 3.9 6.5 5.7 2.3 3.5 6.4 5.0 5.5 3.5 3.4 4.8 5.6 4.8 4.6 3,5 2.5 2.7
Pro
10.9 9.9 8.1 8.9 12.5 11.5 12.3 11.7 8.9 15.6 8.7 9.7 9.6 10.8 9.6 7.5 9.5 7.5 11.1
GIy
6.5 9.6 10.0 8.6 9.5 5.5 6.9 8.6 7.7 10.6 5.2 9.0 8.6 12.3 7.5 7.8 9.3 8.7 7.1
Ala
7.3 6.3 6.5 6.9 6.8 8.0 5.6 5.2 5.7 3.9 7.5 5.4 6.7 6.1 7.7 6.9 6.9 6.0 5.7
Val
4.3 4.2 3.6 3.6 4.3 5.2 6.4 7.7 4.3 2.6 5.0 5.5 4.5 3.0 5.6 4.7 2.5 3.4 3.9
Ile
Leu
7.1 8.2 5.5 8.6 5.8 6.6 7.3 6.8 7.4 3.1 4.9 9.3 6.2 6.4 5.7 7.0 7.4 10.3 8.3
AMINO ACID COMPOSITIONS OF THE ISOLATED PROTEINS OF THE LARGE SUBUNIT OF RAT LIVER RIBOSOMES
TABLE III
2.9 2.0 5.9 2.5 2.0 2.2 1.7 0.9 1.8 1.5 2.8 3.4 1.8 1.9 1.8 2.7 3.0 1.9 2.3
Tyr
4.0 2.5 3.7 2.2 3.0 2.8 3.8 2.P 2.3 2.0 2.8 1.3 2.4 2.4 2.6 2.9 1.6 1.6 2.8
Phe
2 3 4 2 3 2 3 3 2 2 3 2 2 1 5 2 2 2 3
Number o f estimations
bo Cn
426 nificant differences with respect to some amino acids, when comparing the individual proteins with one another. The ribosomal proteins contain a high amount of charged amino acids. The proteins of the small ribosomal subunit contain on the average 21 mol% basic and 20 mol% acidic amino acids. In the proteins of the large subunit 23 mol% basic and 18 mol% acidic amino acids are found. The amount of amidation was not determined. Therefore, the positive net charge may be even higher than estimated from the amino acid composition. Nevertheless, the electrophoretic mobility of the ribosomal proteins at pH 8.3 is correlated to some extent with their content of charged amino acids. Some proteins, moving faster in the first dimension (e.g. $20, $26, $27, S31, L27, L33, L34) contain a higher amount of basic amino acids than acidic ones. On the other hand, slowly moving proteins (e.g. $23, L8, L13, L14, L24) contain more acidic amino acids. Discussion
The availability of pure ribosomal proteins is an important prerequisite for investigations of the structure and function of ribosomes [17]. However, the preparation of pure proteins from eukaryotic ribosomes is rather difficult. Until now only Westermann et al. [18,19] and Collatz et al. [20] have published the isolation of proteins of the small subunit of rat liver ribosomes by a combination of several chromatographic procedures. In the same way preparation of proteins of the large subunit has been performed by Tsurugi et al. [21, 22]. In this paper we describe the preparation of proteins of both subunits of rat liver ribosomes by a two
427
amino acids [21,22]. The differences in the amino acid compositions found for other proteins may be due mainly to different procedures used for their isolation and thus to different degrees of intactness and purity. Differences in the conditions of protein hydrolysis and in the amino acid determinations may further contribute to a certain extent to the observed differences in the amino acid compositions estimated so far.
Acknowledgements We thank Mrs. S. Gross for skilful technical assistance and Mrs. D. Fiedler and Mr. R. Dettmer for performing the amino acid analyses.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Kaltschmidt, E. and Wittmann, H.G. (1970) Anal. Biochem. 36,401--412 Welfle, H., Stahl, J. and Bielka, H. (1972) FEBS Lett. 26,226--232 Welfle, H., Goerl, M. and Bielka, H. (1978) Mol. Gen. Genet., in p r e s s Howard, G.A. and Traut, R.R. (1973) FEBS Lett. 2 9 , 1 7 7 - - 1 8 0 Henkel, B., Welfle, H. and Bielka, H. (1974) Acta Biol. Med. Germ. 3 3 , 6 9 1 - - 6 9 8 Blobel, G. and Sabatini, D. (1971) Proc. Natl. Acad. Sci. U.S. 6 8 , 3 9 0 - - 3 9 4 Eikenberry, E.F., Bickle, T ~ , Traut, R.R. and Price, C.A. (1970) Eur. J. Biochem. 12, 113--116 Martini, O.H.W. and Gould, H,I. (1971) J. Mol. Biol. 62, 403--405 Lastick, S.M. and McConkey, E.H. (1976) J. Biol. Chem. 251, 2867--2875 Welfle, H. (1971) Acta Biol. Med. Germ. 27,547--551 Devenyi, Y. (1969) Aeta Bioehim. Biophys. Acad. Sei. Hung. 4, 297--299 Houston, L.L. (1971) Anal. Biochem. 44, 81--86 Lees, M.B. and Paxman, S.A. (1973) J. Neurochem. 21, 1031--1034 Stein, S., Chang, C.H., B6hlen, P., Imal, K. and Udenfriend, S. (1974) Anal. Biochem. 6 0 , 2 7 2 - - 2 7 7 Canfield, R.E. (1963) J. Biol. Chem. 238, 2696--2707 Smyth, D.G., Stein, W.H. and Moore, S. (1963) J. Biol. Chem. 239,227--234 Wittmann, H.G. (1976) Eur. J. Blochem. 61, 1--13 Westermann, P. and Bielka, H. (1973) Molee. Gen. Genet. 126, 349--356 Westennann, P. and Bielka, H. (1974) Acta Biol. Med. Germ. 33,531---537 Collatz, E., Wool, I.G., Lin, A. and St0ffler, G. (1976) J. Biol. Chem. 251, 4666--4672 Tsurugi, K., Collatz, E., Wool, I.G. and Lin, A. (1976) J. Biol. Chem. 251, 7940--7946 Tsunlgi, K., Collatz, E., Todokoro, K. and Wool, I.G. (1977) J. Biol. Chem. 252, 3961--3969
Biochimica et Biophysica Acta, 519 (1978) 418--427 © Elsevier/North-Holland Biomedical Press
BBA 99188
PREPARATIVE TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS OF RAT LIVER RIBOSOMAL PROTEINS AND DETERMINATION OF THEIR AMINO ACID COMPOSITIONS *
M. GOERL, H. WELFLE and H. BIELKA
Central Institute o f Molecular Biology of the Academy of Sciences of the G.D.R., Department o f Cell Physiology, 1115 Berlin-Buch (Germany) (Received September 26th, 1977) (Revised manuscript received December 20th, 1977)
Summary 1. By enlarging the dimensions of the gels used in the'usual analytical twodimensional polyacrylamide gel electrophoresis it is possible to separate much larger amounts of ribosomal protein in comparison to analytical separations. 15 mg of protein mixture of small or large subunits of rat liver ribosomes can be separated by this procedure. 2. The positions of the proteins in the two~limensional patterns are identified with a special staining procedure. The proteins are eluted from the gels with SDS/phosphate buffers. 3. The purity of the extracted proteins was tested by one-dimensional SDSpolyacrylamide gel electrophoresis and two
Introduction The two
* Paper No. XXVI o f t h e series: Proteins o f animal ribosomes. Abbreviation: SDS, s o d i u m d o d e c y l sulphate.
419 were used in the analytical two
Preparation of ribosomal proteins. Rat liver ribosomes were prepared from the postmitochondrial supernatant of homogenates of rat liver as described earlier [5]. The crude ribosomes were dissociated essentially according to Blobel and Sabatini [6] by incubation with puromycin in the presence of 5 mM Tris/ 500 mM KC1/1 mM MgC12/0.5 mM ZnC12/10 mM ~-mercaptoethanol, pH 7.7. The subunits were separated on a large scale by zonal centrifugation according to Eikenberry et al. [7] using the B15 titanium rotor (Beckman) as already described [5]. The ribosomal proteins were extracted from the subunits with 0.25 M HC1, precipitated with acetone and stored at --20°C [2]. Two-dimensional polyacrylamide gel electrophoresis on preparative scale. Compositions of gels and buffers are the same as in the analytical two
420 buffer (in each case 5 1 100 mM sodium phosphate/0.1% SDS, pH 7.1) was changed after 24, 72 and 96 h. The buffer circulated between the electrode chambers during the run. 2-D System C. In the first dimension we used gel composition and buffers as described by Lastick et al. [9] (4% gels, pH 8.6). In the second dimension the electrophoretic conditions were the same as described for 2-D system A. Localisation of the single proteins in the gel slab. For localisation of the proteins the gels were stained either in a thin slab cut from the gel slab or at the surface of one side of the gel slab with 0.01% Coomassie Brilliant Blue G 250 in 12% trichloroacetic acid. Extraction of the proteins. Gel pieces containing the proteins were crushed and dialysed against different buffers in the following manner: (1) 100 mM sodium phosphate, pH 7.1/0.05% SDS/10 mM ~-mercaptoethanol (3 X 500 ml, 20 h); (2) 10 mM sodium phosphate, pH 7.1/ 0.01% SDS/10 mM ~-mercaptoethanol (3 X 500 ml, 6 h); (3) 1 mM sodium phosphate, pH 7.1/0.005% SDS/10 mM ~-mercaptoethanol (3 X 500 ml, 20 h). After dialysis the solutions were filtered and lyo'phflized. Characterisation of the isolated proteins by one-dimensional SDS electrophoresis. Gels and electrophoresis buffer were of the same composition as the gels and buffers used in the second dimension of the system B. Gels of 8 cm length and 6 mm diameter were used. An aliquot of the lyophilized material (about 5%) was resolved in 40/~l 10 mM sodium phosphate, pH 7.1/8 M urea/l% SDS/ 1% ~-mercaptoethanol at room temperature for 2 h. Bromophenol blue was added to the samples as marker dye and 20 ttl of the protein solution were applied to the gels. Electrophoresis was performed vor 6 h at 45 V. The proteins were stained with Coomassie Brilliant Blue and scanned at 600 nm with the Gilford 2400-2 spectrophotometer. Test of purity of the isolated proteins by two-dimensional electrophoresis. The lyophilized proteins were dissolved in 0.6 ml 0.25 N HC1/10 mM ~-mercaptoethanol in each case and 50 ttg bovine serum albumin were added. The proteins were precipitated with 5% trichloroacetic acid, washed at first with 5% trichloroacetic acid/10 mM ~-mercaptoethanol and then several times with acetone. The proteins were dissolved in 50 ttl 8 M urea/10 mM ~-mercaptoethanol at 20°C overnight. The proteins were reduced by incubation with 5 ttl 10 mM 1,4
421 Results
Separation and purity of the proteins The two
422
2
4
lip
,w
9
8
10
12
qlB
qlD
14
15a
16 q
18
~i
24 / 25 Q
28
29
Fig. 1. A n a l y t i c a l 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 s i s o f i s o l a t e d r i b o s o m a l P r o t e i n s o f t h e s m a l l s u b u n i t . 1 0 - # g p r o t e i n p o r t i o n s w h i c h w e r e t z e a t e d w i t h a c e t o n e t o r e m o v e SDS w e r e a n a l y z e d in t h e 2-D s y s t e m A . ~ n ~ a n e q u i p m e n t f o r a n a l y t i c a l s e p a r a t i o n s a t 1 3 0 V f o r 4 h in t h e first a n d 4 0 V a n d 16 h in t h e s e c o n d d i m e n s i o n . T h e P r o t e i n s w e r e s t a i n e d w i t h C o o m a s a t e Brilliant Blue R 2 5 0 .
423
Amino acid composition of the proteins separated by preparative two-dimensional electrophoresis. As known from the literature [12--14], proteins isolated by polyacrylamide gel electrophoresis are suitable for the determination of their amino acid compositions. In order to check whether also the preparative two~limensional electrophoresis procedure used by us is appropriate to obtain correct values for the amino acid composition of proteins after two~iimensional electrophoresis, we first of all analyzed the amino acid compositions of lysozyme and ribonuclease, extracted from the gels slabs after two~limensional electrophoresis. In Table I, the corresponding values for both enzymes, untreated as well as after twodimensional electrophoresis, are given and compared with values known from the literature [15,16]. With the exception of proline and isoleucine, the values are in fairly good agreement. This demonstrates that proteins prepared by two-dimensional electrophoresis in polyacrylamide gel are well suitable for the determination of their amino acid composition. Probes of polyacrylamide taken from 2-D gel slabs, dialyzed, lyophilized and hydrolyzed under the same conditions as for amino acid analysis did not reveal any ninhydrin positive material in the positions of the amino acids. In Tables II and III the amino acid compositions of single ribosomal proteins prepared by two~limensional electrophoresis are demonstrated. The amino acid compositions of the ribosomal proteins from rat liver are fairly similar, as described already in former papers [18--20]. On the other hand, there are sigTABLE I AMINO ACID COMPOSITION OF UNTREATED AND ELECTROPHORETICALLY ZYME AND RIBONUCLEASE A IN COMPARISON TO THE LITERATURE
PREPARED LYSO-
T h e values are e x p r e s s e d in m o l / 1 0 0 t o o l a m i n o acids, M e t , Cys a n d Trp w e r e n o t d e t e r m i n e d , b u t t a k e n i n t o a c c o u n t for c o r r e c t i o n o f o u r data. T h e value o f L e u is so l o w b e c a u s e o f t h e short h y d r o l y s i s t i m e o f 1 6 h. n.d., n o t d e t e r m i n e d . Lysozyme ref. 1 5
Lys H~ A~ A~ Thr Ser G~ Do Gly Ah Cys Val Met He Leu Tyr Phe ~p
Ribonuclease Untreated
Electrophoretically prepared
ref. 16
8.1 3.2 3.2 12.1
Untreated
8.2 3.0 3.4 12.1
Electrophoretically prepared
4.7 0.8 9.5 16.3
4.9 0.9 8.8 15.3
4.3 0.9 8.0 14.9
7.7 3.1 3.2 12.6
5.4
5.2
5.3
8.1
7.7
8.4
7.8
7.2
8.2
12.1
11.1
12.3
3.9 1.6 9.3 9.3 6.2 4.7 1.6 4.7 6.2 2.3 2.3 4.7
4.3 3.7 9.1 8.9 n.d. 4.2 n.d. 3.7 6.4 2.5 2.6 n.d.
4.5 3.3 9.7 8.5 n.d. 4.2 n.d. 3.7 6.0 2.7 2.5 n.d.
9.7 3.2 2.4 9.7 6.5 7.3 3.2 2.4 1.6 4.8 2.4 0
9.7 5.9 2.8 9.4 n.d. 6.5 n.d. 1.2 1.8 5.1 2.4
9.6 3.6 2.4 9.7 n.d. 6.8 n.& 1.4 2.0 5.1 2.4
--
--
Lys
8.6 8.6 11.8 10.2 12.7 13.4 11.1 11.4 9.3 8.0 12.2 8.3 7.8 9.7 9.8 7.3 9.7 5.8 17.6 9.7 9.5 4.6 6,2 12.5
Protein
$1 2 3 4 7 9 10 11 12 13 14 16 17 18 20 21 22 23 26 27 28 29 30 31
1.8 1.7 2.1 2.8 2.4 2.0 1.9 2.5 2.3 4.0 2.1 1.7 2.4 3.8 2.9 3,1 2.7 2.6 1.5 2.5 4.7 0.7 3.0 2.0
His
5.3 7.3 6.3 9.1 5.6 12.7 7.0 10.4 12.2 9.6 8.2 8.8 8.8 10.8 9.3 7.8 8.0 8.0 5.8 12.8 6.5 14.7 7.9 6.6
Arg
6.8 6.8 10.1 10.4 11.2 7.7 8.1 8.8 8.8 8.4 8.6 10~ 7.9 6.8 8.5 10.2 7.7 14.1 10.9 9.5 6.2 8,2 8.6 7.6
Asp
7.2 5.8 6.1 7.4 7.0 5.2 4.2 4.9 3.4 5.1 7.2 7.0 6.2 4.9 4.8 4.7 9.5 3.0 4.2 2.5 7.6 9.3 3.2 4.9
Thr
T h e values are e x p r e s s e d in t o o l / 1 0 0 m o l a m i n o acids.
6.4 5.7 5.6 4.4 6.0 5.8 7.4 6.5 5.5 5.6 5.3 5.9 8.2 6.3 6.0 8.1 7.9 10.3 7.8 5.6 8.6 7.8 11.6 13.2
Set
8.3 12.1 12.0 7.6 11.6 11.9 13.7 11.7 11.4 10.2 11.0 13.0 11.4 12.1 11.0 11.5 11.1 9.9 7.5 8.1 12.9 14.1 11.9 10.5
Glu
5.4 5.4 3.9 4.9 2.4 4,3 4.3 3.5 3.4 3.3 4.2 4.3 4.4 4.9 3.6 3.1 6.0 1.8 4.6 9.6 6.3 3.1 2.6 4.0
Pro
16.1 11.1 9.5 9.8 10.9 7.2 7.5 11.9 10.4 14.3 9.8 9.2 13.1 9.5 12.2 12.2 8.3 12.4 10.9 7.8 9.5 9.0 17.8 16.6
GIy
9.4 7.6 6.6 6.1 5.9 6.9 6.1 6.1 6.0 7.6 5.4 5.7 9.7 5.3 9.8 6.5 5.8 10.7 9.0 11.5 5.1 3.9 6.6 9.0
Ala
7.0 9.0 8.9 5.7 7.2 6.1 7.9 4.9 6.7 5.7 6.6 7.7 5.5 6.2 8.1 6.6 7.1 5.6 5.5 8.0 4.8 10.5 3.9 5.7
Val
5.1 5.3 4.7 7.8 5.0 4.1 5.9 4.8 4.8 3.8 6.1 6.4 4.4 4.4 2.2 4.7 7.6 5.7 3.0 4.4 2.4 3.4 3.9 0.9
Ile
8.0 8.8 7.9 8.5 7.3 9.5 9.3 8.5 10.8 8.0 6.8 7.5 6.8 9.4 7.9 8,1 6.4 4.0 8.7 4.0 8.4 8.8 6.6 2.8
Leu
AMINO ACID COMPOSITIONS OF THE ISOLATED PROTEINS OF THE SMALL SUBUNIT OF RAT LIVER RIBOSOMES
T A B L E II
1.3 1.5 1.1 1.3 1.0 1.7 0.6 1.9 1.7 2.5 2.0 1.9 1.0 2.1 1.2 1.1 0.4 1.6 1.7 1.7 3.6 0.6 2.5 1.2
Tyr
3.8 3.3 3.7 4.1 3.5 2.0 5.2 2.4 3.3 3.9 4.2 3.0 2.7 3.7 2.9 4.6 2.0 4.4 1.7 2.6 4.1 1.8 3.6 2.6
Phe
2 3 2 2 4 2 5 3 4 2 3 4 2 3 2 1 4 3 2 2 1 2 1 1
Number of estimations
bo
L1 2 3 4 7 8 13 14 16 24 25 26 27 29 30 31 33 34 38
tein
Pro-
10.0 10.0 11.1 12.7 10~ 9.3 7.9 7.4 11.9 10.0 13.9 12.0 9.2 10.0 11.7 13.1 12.6 17.1 13.6
Lys
3.5 2.1 1.8 2.3 3.2 2.9 2.2 2.9 2.9 2.0 3.2 2.2 2.3 2.6 2.1 2.9 1.8 1.5 1.8
His
7.7 9.0 9.2 8.4 9.9 5.8 8.2 5.7 8.0 5.1 10.4 6.0 10.6 7.4 9.2 10.8 12.5 11.1 5.7
Arg
8.4 8.5 11.3 6.3 7.3 9.8 9.2 11.2 7.8 6.2 9.2 8.2 8.6 8.9 10.8 9.0 5.1 6.2 8.4
Asp
6.5 5.6 4.3 5.8 4.7 5.6 4.8 5.4 4.7 4.1 4.1 5.9 6.3 3.3 6.1 4.0 6.0 5.1 4.7
Thr
T h e v a l u e s are e x p r e s s e d in t o o l / 1 0 0 t o o l a m i n o acids.
6.5 6.9 4.0 6.9 5.8 9.5 6.8 7.6 7.0 14.0 8.2 7.9 9.7 8.1 6.1 6.7 8.4 5.8 11.0
Set
10.7 9.6 11.3 9.9 9.3 12.5 13.3 10.7 14.5 13.9 11.6 10.6 8.9 11.2 8.7 9.3 9.7 10.8 10.9
Glu
3.9 5.6 3.9 6.5 5.7 2.3 3.5 6.4 5.0 5.5 3.5 3.4 4.8 5.6 4.8 4.6 3,5 2.5 2.7
Pro
10.9 9.9 8.1 8.9 12.5 11.5 12.3 11.7 8.9 15.6 8.7 9.7 9.6 10.8 9.6 7.5 9.5 7.5 11.1
GIy
6.5 9.6 10.0 8.6 9.5 5.5 6.9 8.6 7.7 10.6 5.2 9.0 8.6 12.3 7.5 7.8 9.3 8.7 7.1
Ala
7.3 6.3 6.5 6.9 6.8 8.0 5.6 5.2 5.7 3.9 7.5 5.4 6.7 6.1 7.7 6.9 6.9 6.0 5.7
Val
4.3 4.2 3.6 3.6 4.3 5.2 6.4 7.7 4.3 2.6 5.0 5.5 4.5 3.0 5.6 4.7 2.5 3.4 3.9
Ile
Leu
7.1 8.2 5.5 8.6 5.8 6.6 7.3 6.8 7.4 3.1 4.9 9.3 6.2 6.4 5.7 7.0 7.4 10.3 8.3
AMINO ACID COMPOSITIONS OF THE ISOLATED PROTEINS OF THE LARGE SUBUNIT OF RAT LIVER RIBOSOMES
TABLE III
2.9 2.0 5.9 2.5 2.0 2.2 1.7 0.9 1.8 1.5 2.8 3.4 1.8 1.9 1.8 2.7 3.0 1.9 2.3
Tyr
4.0 2.5 3.7 2.2 3.0 2.8 3.8 2.P 2.3 2.0 2.8 1.3 2.4 2.4 2.6 2.9 1.6 1.6 2.8
Phe
2 3 4 2 3 2 3 3 2 2 3 2 2 1 5 2 2 2 3
Number o f estimations
bo Cn
426 nificant differences with respect to some amino acids, when comparing the individual proteins with one another. The ribosomal proteins contain a high amount of charged amino acids. The proteins of the small ribosomal subunit contain on the average 21 mol% basic and 20 mol% acidic amino acids. In the proteins of the large subunit 23 mol% basic and 18 mol% acidic amino acids are found. The amount of amidation was not determined. Therefore, the positive net charge may be even higher than estimated from the amino acid composition. Nevertheless, the electrophoretic mobility of the ribosomal proteins at pH 8.3 is correlated to some extent with their content of charged amino acids. Some proteins, moving faster in the first dimension (e.g. $20, $26, $27, S31, L27, L33, L34) contain a higher amount of basic amino acids than acidic ones. On the other hand, slowly moving proteins (e.g. $23, L8, L13, L14, L24) contain more acidic amino acids. Discussion
The availability of pure ribosomal proteins is an important prerequisite for investigations of the structure and function of ribosomes [17]. However, the preparation of pure proteins from eukaryotic ribosomes is rather difficult. Until now only Westermann et al. [18,19] and Collatz et al. [20] have published the isolation of proteins of the small subunit of rat liver ribosomes by a combination of several chromatographic procedures. In the same way preparation of proteins of the large subunit has been performed by Tsurugi et al. [21, 22]. In this paper we describe the preparation of proteins of both subunits of rat liver ribosomes by a two
427
amino acids [21,22]. The differences in the amino acid compositions found for other proteins may be due mainly to different procedures used for their isolation and thus to different degrees of intactness and purity. Differences in the conditions of protein hydrolysis and in the amino acid determinations may further contribute to a certain extent to the observed differences in the amino acid compositions estimated so far.
Acknowledgements We thank Mrs. S. Gross for skilful technical assistance and Mrs. D. Fiedler and Mr. R. Dettmer for performing the amino acid analyses.
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