Delaunay, J.

Huynh-Van-Tan, 

Schapira, G.

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Volume 17, number 1 FEBS LETTERS Sqxtfmnbex 1971 
EUKARYOTIC RIBOSOMAL PROTEINS. 
TWO-DIMENSIONAL ELECTROPHORETIC STUDIES 
HUYNH-VAN-TAN, J. DELAUNAY and G. SCHAPIRA 
Institut de Pathologie iUol&daire* 
24, ruedu Faubourg Saint-Jacques, Paris 14e, France 
Received 14 June 197 1 
I. Introduction 
Although some of the ribosomal proteins are 
believed to be different from species to species, they 
are thought to be identical within any one species 
[l-6]. It has also been reported that ribosomzs, even 
from the same tissue,-are heterogeneous in their pro- 
tein content and/or function [7-131. As for the num- 
ber of proteins in an eukaryotic ribosome, it has been 
estimated to be similar [ 141 or higher [ 15 ] than in 
prokaryotic ribosomes. 
Evidence is reported below which indicates, as ex- 
pected, that ribosomes from two species differ in 
some of their protein components. But unexpectedly, 
some differences are also found in two different organs 
in the same species. Furthermore eukaryotic ribosomal 
proteins are shown to be more numerous than pro- 
karyotic ones. 
2. Experimental 
2.1. Preparation of rabbit reticulocyte ribosomes 
The ribosomes obtained by centrifugation [ 16- 1 S] 
are suspended in (one-tenth of initial volume) buffer 
A (0.01 M tris-acetate, pH 7.2; 5 X 10m3 M Mg acetate; 
0.3 M KCl). The ribosomes (20 to 25 mg of RNA 
estimated by the ratio of absorbance at 2600 A and 
2800 A) are layered on a 1 X 20 cm column of DEAE 
* Institut d’Universit6, Groupe U 15 de I’Institut National de 
la Sank ct dc la Recherche Mkdicale, Laboratoirc associk au 
Centrc National dc la Recherche Scicntitiquc. 
cellulose (Whatman DE 11) regenerated by 0.2 M 
NaOH and 1 M HCl, and equilibrated by buffer A. The 
column is thoroughly washed free of hemoglobin and 
other proteins until the absorbance is back to the 
initial value. 
The ribosomes are eluted by buffer B (0.01 M 
tris-acetate pH 7.2; 5 X 10m3 M Mg acetate; 0.55 M 
KCl), and collected by centrifugation at 150,000 g, 
90 min [19-211. 
2.2. Preparation of ribosomes from rabbit liver and 
rat liver 
50 g of liver are homogeneized in 100 ml of buffer 
A [ 221. Another 100 ml of buffer A is added and 
membranes are separated by centrifugation at 17,000 
g, 10 min. The supernatant is centrifuged again 
(30,000 g, 60 min) to separate glycogen and membrane 
debris. Ribosomes are obtained by centrifugation of 
the supernatant at 150,000 g, 90 min. They are sus- 
pended in buffer A (one-tenth of initial volume). Ribo- 
somal aggregates are eliminated by centrifugation 
30,000 g, 60 min. As described above, the supernatant 
is layered on a DEAE cellulose column which is then 
thoroughly washed free of ferritin and other proteins 
with buffer A; the ribosomes are eiuted with buffer 
B and collected by centrifugation. The use of sodium 
deoxycholate, which damages ribosomes [ 231 is 
avoided. 
However, another method using sodium deoxy- 
cholate and described elsewhere [29J has been used to 
see whether the number of ribosomal proteins is iden- 
tic al. 
North-Holland Publishing Company - Amsterdam 163 
Volume 17, number 1 FEBS LETTERS September 1971 
2.3. Preparation of ribosomal proteins 3. Results and discussion 
The proteins are extracted by 2 M LiCl dialyzed 
against water and lyophilized [24,25] The DOC 
ribosome pellet is treated overnight at 0” with 6 M 
urea and 4 M LiCI; after centrifugation (27,000 g, 15 
min) the supernatant is dialyzed overnight against a 
large volume of 0.01 M HCl, the centrifuged and the 
supernatant, containing the ribosomal protein, is 
finally lyophilized. 
3.1. Number of proteins in an eukaryotic ribosome 
The number of spots on the three electrophoretic 
gels indicates that the number of proteins in eukaryo- 
tic ribosomes purified on DEAE cellulose is higher 
than that in prokaryotic ribosomes [27]. 7.5 spots 
can be counted for rabbit liver ribosomes (fig. 2). 
This number may vary a little depending on the origin 
of the ribosome (see below) and the method of pre- 
paration. The possibility of a spot being an extra- 
ribosomal artifact may not be entirely excluded. 
However, the possibility is not very likely from the 
following considerations: 
2.4. Electrophoresis 
Electrophoresis and staining of 1 or 2 mg of pro- 
teins are carried out according to Kaltschmidt and 
Wittmann [27] except for the following temperature 
during the run: 4” ; first dimension: acrylamide con- 
centration 4% pH 8.6 (constant voltage 200 V, initial 
intensity 5 mA/tube, run time: 14 hr); second dimen- 
sion: acrylamide concentration 9% pH 4.6 (constant 
voltage 90 V, initial intensity 100 mA/gel, run time 
17 hr. 
(1) Ribosomes purified on DEAE cellulose already 
loose about 30% of their protein content compared 
with washed ribosomes [20] 
(2) A spot may be the result of an aggregation of 
two proteins, or conversely be derived from another 
one. This possibility is not very likely: it was observed 
in one only case in E. coli ribosomal proteins 1281. 
(3) Ribosomal proteins from one tissue (liver) pre- 
pared by two different methods, despite some dif- 
Fig. 1. 
164 
Ribosomal proteins from rat liver: Two dimensional polyacrylamide gel electrophoresis according to the Kaltschmidt 
Wittmann’s methods. 
and 
Volume 17, number 1 FEBS LETTERS September 1971 
+ 
Fig. 2. Ribosomal proteins from rabbit liver. 
Fig. 3. Ribosomal proteins from rabbit reticulocytes. 
165 
Volume 17, number 1 FEBS LETTERS September 1971 
ferences, both show a number of proteins higher than 
in E. coli. 
(4) Ribosomal proteins from different tissues pre- 
pared by the same method also show a number of pro- 
teins higher than in E. coli. 
(5) Ribosomal proteins from normal liver and 
hepatomas show no differences between themselves, 
although the hepatoma synthesizes many proteins 
different from normal liver [29] . 
So even in the case of artifacts which would slightly 
change the exact number of proteins in eukaryotic 
ribosomes, this would not change the conclusion that 
eukaryotic ribosomal proteins are more numerous than 
prokaryotic ones. This conclusion agrees with Warner’s 
who estimated the number of ribosomal proteins in 
yeast to be 80 [1.5]. 
3.2. Comparison between ribosomal proteins from 
rabbit liver and rat liver (figs. 1 and 2) 
As expected from earlier studies [ 1, 131 the lectro- 
phoretic patterns show some differences. The better 
resolution of the two-dimensional electrophoresis 
t 
further shows that the g neral pattern is very similar 
and that most ribosomal proteins behave similarly, a 
fact which cannot be observed with one-dimensional 
disc electrophoresis. 
3.3. Comparison between ribosomal proteins from 
rabbit reticulocytes and liver 
The ribosomal protein patterns from these two 
tissues also shows some differences (figs. 2,3 and 4). 
The hepatic cell synthesizes many different proteins 
which may result in the observed supplementary spots, 
however the probability of extra-ribosomal contamin- 
ation is very low as discussed above. 
This criticism does not hold for the reticulocytes 
which synthesize mostly QI and fl chain of hemoglobin. 
On a separate electrophoretic run (Y chain, /I chain, 
globin and hemoglobin are shown not to migrate to 
the loci of the observed differences, They migrate 
into the left half of the gel. As far as the proteins 
being synthesized upon the ribosomes are concerned, 
their different lengths would make them invisible on 
the gel. The extra spots from reticulocytes ribosomes 
cannot therefore be considered as contaminants,. 
+ - 
- 
I’ig. 4. Composite figure from fire 2 and 3, showing the observed differences. l Spots common to rabbit liver and reticulocytes. 
o Spots found only in rabbit liver. c Spots found only in rabbit reticulocyte. 
166 
Volume 17, number 1 FEBSLE’ITERS September 1971 
Di Girolamo and Cammarano [l] found no differ- 
ence between rabbit reticulocyte and rabbit liver ribo- 
somal proteins but they did show that the small sub- 
unit from these two tissues present different protein 
patterns. However they considered that the differen- 
ces should be artifacts and may be due t’o some un- 
known enzymatic properties of the separated small 
subunits. Further investigation would probably show 
that the differences that we observed lie in the small 
subunit as found by Di Girolamo. 
Acknowledgements 
This work was supported by grant from “DClBga- 
tion GMrale ?I la Recherche Scientifique et Tech- 
nique et ses Comit& Scientifiques (Fonds de d&e- 
loppement)“. 
References 
[l] M. Di Girolamo and P. Cammarano, Biochim. Biophys. 
Acta 168 (1968) 181. 
[ 21 V. Mutolo, G. Giudic, V. Hopps and G. Donatuti, Bio- 
chim. Biophys. Acta 138 (1967) 214. 
[ 31 J.P. WalIer, J. Mol. Biol. 10 (1964) 544. 
[4] E. Otaka, T. Itoh and S. Osawa, J. Mol. Biol. 33 (1968) 
93. 
[S] M. Takagi, T. Tanaka and K. Ogata, Biochim. Biophys. 
Acta 217 (1970) 148. 
[6] R.B. Low and LG. Wool, Science 155 (1967) 330. 
[7] E.R. Burka and S.I. Bulova, Biochem. Biophys. Res. 
Commun. 42 (1971) 801. 
181 
[91 
1101 
1111 
[121 
[131 
[141 
1151 
(161 
[171 
1181 
[191 
I201 
1211 
I221 
[231 
1241 
1251 
WI 
(271 
VI 
~291 
CM. Redman, J. Biol. Chem. 244 (1969) 4308. 
S.J. Hicks, J.W. Drysdale and H.N. Munro, Science, 164 
(1969) 584. 
G.C. Priestly, M.L. Pruyn and R.A. Malt, Biochim. 
Biophys. Acta 190 (1969) 154. 
P. Sickevitz and G.E. Palade, J. Biophys. B&hem. 
Cytol. 7 (1960) 619. 
M.C. Ganoza and C.A. Williams, Proc. Natl. Acad. Sci, 
U.S. 63 (1969) 1370. 
A.T. Bui and G. Schapira, C.R. Acad. Sci. (Paris) 264 
(1967) 2417. 
H.J. Gould, Nature 227 (1970) 1145. 
J.R. Warner, J. Biol. Chem. 246 (1971) 447. 
J. Kruh, J.C. Dreyfus and G. Schapira, Biochim. Bio- 
phys. Acta 55 (1962) 690. 
H. Borsook, C.L. Deasy, A.J. Haagen-Smit, G. Keighley 
and P. Lowy, J. Biol. Chem. 196 (1952) 669. 
J. Kruh, J. Rosa, J.C. Dreyfus and G. Schapira, Biochim. 
Biophys. Acta 49 (1961) 509. 
M. Salas, A.M. Smith, W.M. Stanley, A.J. Wahba and 
G. Ochoa, J. Biol. Chem. 240 (1965) 3988. 
A.V. Furano, J. Biol. Chem. 241 (1966) 2237. 
E.A. Peterson and E.L. Kuff, Biochemistry 8 (1969) 
2916. 
M.S. Pollack, G. Schapira and J.C. Dreyfus, Bull. Sot. 
Chim. Biol. 52(1970) 891. 
E.R. Burka, Biochim. Biophys. Acta 145 (1967) 506. 
J.B. Curry and R.T. Hersh, Biochem. Biophys. Res. 
Commun. 6 (1961/62) 415. 
A.P. Mathias and R. Williamson, J. Mol. Biol. 9 (1964) 
498. 
P. Spitnik-Elson, Biochem. Biophys. Res. Commun. 18 
(1965) 557. 
E. Kaltschmidt and H.G. Wittmann, Anal. Biochem. 30 
(1969) 132. 
E. Kaltschmidt and H.G. Wittmann, Proc. Natl. Acad. 
Sci. U.S. 67 (1970) 1276. 
J. Delaunay, F. Schapira and G. Schapira, to be 
published. 
167 


Eukaryotic ribosomal proteins. Two-dimensional electrophoretic studies 

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Eukaryotic ribosomal proteins. Two-dimensional electrophoretic studies

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