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The distribution of protein antigens among Escherichia coli ribosomes
330
P R E L I M I N A R Y NOTES
I F. H. WILT AND T. I-tULTIN, Biochem. Biophys. Res. Commun., 9 (1962) 313 . * A, C. GRIFFINANDM. A. O'NEAL, Biochim. Biophys. Acta, 61 (1962) 469 . * H. I~..V. ARNSTEIN, R. A. COX AND J. A. HUNT, Nature, 194 (1962) lO42. 4 j. G. FLAKS, E. C. COX AND J. R. \VHITE, Biochem. Biophys. Res. Commun., 7 (1962) 385. $ S. H. BARONDESAND M. W. NIRENBERG,Science, 138 (1962) 81o. Received J a n u a r y 3rd, 1963 Biochim. Biophys..4cta, 68 (1963) 328-33o
PN 6086 The distribution of protein antigens among Escherichia coli ribosomes In previous communications 1-4 we have presented qualitative as well as quantitative evidence defining the antigenic community existing between ribosomes of various species at the level of R N A for "distant heterologous ribosomes", and at the RNA and protein level for "neighbouring heterologous ribosomes". Homologous ribosomes possess in addition to the two aforementioned types, specific antigens of a protein nature. In order to assess whether these ribosomal protein antigens were present on all ribosomes or restricted to certain, goat-serum anti-ribosomes of Escherichia coli KI2 3000 (obtained b y twice weekly intravenous inlections, each of 6 mg of ribosomes) was therefore fractionated into its component antibodies by complete successive absorptions with the ribosomes of Welchia per/ringens, Alcaligenes /aecalis, Proteus vulgaris, E. coli K12 3000 Del X74 and E. coli K~2 3300. To the antibodies recuperated (as described below, from the specific precipitates formed successively with the ribosomes of Welchia per/ringens, Alcaligenes /aecalis, and Proteus vulgaris, were then added the ribosomes of E. coli K12 3000. In another series of experiments, E. coli ribosomes were added to the antibodies remaining in the supernatant after complete absorption by the ribosomes of Proteus vulgaris, E. coli K~2 3000 Del X74 and E. coli K12 3300. These precipitation reactions were carried out as previously described s. The RNA content of the precipitate ribosomes was further verified by concomitant estimation of the RIgA remaining in the supernatant. Ribosomes prepared essentially by the technique of TISSIIkRES AND WATSON 5, were purified b y 4 successive cycles of centrifugation in the Spinco preparative centrifuge, and finally suspended in o.oi M magnesium acetate, 0.005 M Tris-HC1 buffer (pH 7-4). The specific precipitates formed on addition of ribosomes to the antiserum were twice washed with a solution containing o.oi M magnesium acetate, o.14 M NaC1, 0.005 M Tris-HC1 buffer (pH 7-5) and then incubated at 37 ° for 18 h in o.14 M NaC1, 0.005 M Tris-HC1 buffer (pH 8.5). After centrifuging the resulting suspension, the antibodies were precipi'tated from the supernatant b y (N'H4)2SO4 to 0.35 saturation at 4 ° and finally solubilised in o.14 M NaC1 (pH 7.5)After each complete absorption of the antiserum b y the ribosomes of the different species indicated above, the excess ribosomes remaining in the supernatan~ were eliminated b y centrifuging the serum in the Spinco preparative centrifuge at IOOOOOXg for 4 h. As shown in Fig. I, with the "antibody fraction Welchia per[ringens", the "antibody fraction Alcaligenes/aecalis" and the "antibody fraction Proteus vulgaris", Biochim. Biophys. Acta. 68 (1963) 330-332
331
PRELIMINARY NOTES "Antibody
100,
8C 9
,
fraction" :
0 = Welchio perfringens X = AIcQligenes foecalis Z~ = P r o t e u s v u l g o r i s
6O
a. 4 0 < ~
20
400
1200 2000 2800 3600 4400 ~ug RNA added to l a g of antibody
Fig.
Serum
antidbosomes Of Kla. 3 0 0 0 absorbed byribosomes of:
40
9 ~8
I
E.Coli
0 = Proteus vulgoris iX =E, ColI K12 3 0 0 0 DelX74 FI =E.Coll Kt2 3 3 0 0
3G
~. 2C < z t~ 1C
200
I
I
I
600 1000 1400 1800 2200 ~Jg RNA added to 1mr of anti-serum Fig.
2
(antibodies recuperated from the corresponding specific precipitates as outlined above) the percentage of RNA precipita ted attains or tends to IOO % under conditions of excess antibody. TO determine whether these three types of antibodies were directed against RNA or protein, similar precipitation reactions were carried out with the ribosomes of Fusi/ormis/usi/ormis a "distant heterologue ''z. The percentage of RNA precipitated was IOO ~o with the "antibody fraction Welchia per/ringens", whilst no precipitation was obtained with the "antibody fraction Alcaligenes /aecalis" and the "antibody fraction Proteus vulgaris". Thus, the latter two antibody fractions are exclusively directed against protein antigens. The lower percentages (Fig. 2) obtained with the antibodies remaining in the supernatant after complete absorption of the goat antiserum by the ribosomes of Proteus vulgaris (A), E. eoli K12 3000 Del X74(B ) and E. coli K12 3300 (C) could not be ascribed to the existence of soluble complexes, since the repetition of these experiments with the addition of rabbit-serum anti-goat v-globulin to the supernatant gave no further precipitation of RN'A. That these antigens (identified as basic proteins in immunoelectrophoresis4) are composite parts of E. coli ribosomes and not independent ribonucleoprotein particles can be deduced from the fact that, while the antibodies corresponding to those antigens are not absorbed by the ribosomes of Proteus vulgaris, yet the "antibody fraction Proteus vulgaris" directed against other ribosomal proteins precipitates IOO % of the ribosomes estimated by means of the Biochim. Biophys, Acta,
68 ( I 9 6 3 ) 3 3 0 - 3 3 2
332
PRELIMINARY NOTES
RNA. Neither does the purification procedure eliminate measurable quantities of these antigens from the ribosomes, since identical precipitation curves are obtained with ribosomes prepared in dissociating medium: o.ooi M magnesium acetate--o.o5 M Tris-HC1 buffer (pH 7.5). We can therefore conclude, t h a t these lower percentages of ribosomal R N A in the precipitates obtained with the antisera (A) (B) and (C) indicate the existence of heterogeneity a m o n g the component particles of a ribosome preparation. An a t t e m p t was therefore m a d e to determine at what stage during the formation of ribosomes these antigens appeared. To an exponential culture of E. coli was added IOO /zg/ml of chloramphenicol and the cells were harvested after I h. The ribosomes from these cells gave with antiserum (C) a slight increase of about 15 % of antibodies as well as R N A precipitated, as compared to ribosomes from untreated cells. Since the ribonucleoprotein accumulating in the presence of chloramphenicol eventually becomes 3o-S particles e,7 this result indicates that the antigens reacting with antiserum (C) are found on at least the 3o-S subunits, but p r o b a b l y not with all, since the percentage increase of the R N A precipitated is relatively small. The main feature of this investigation lies in the heterogenous distribution of the antigens linked to the characteristics of the strain, compared to the homogenous distribution found for the ribosomal common antigens. We are therefore investigating whether the ribonucleoprotein precipitated b y antiserum (C) represents specialised ribosomes in view of the fact t h a t TISSI~RES et al. s with E. coli ribosomes, and LAMFROM AND GLOWACKI9 with the ribosomes of rabbit reticulocytes have found that a similar fraction (5-1o %) of the ribosomes of a population which are resistant to dissociation possess increased incorporating ability. The a u t h o r gratefully acknowledges the kind encouragement and valuable discussions of Dr. E. BARBLr t h r o u g h o u t this investigation. Service du Dr. Wahl,
GERARD QUASH
Institut Pasteur (Paris)
x E. BARBI~,J. PANIJEL, P. CAYEUXAND R. WAHL, Compt. Rend., 249 (1959) 338. I •. BARBU AND J. PANIJEL, Compt. Rend., 25° (196o) 1382. * E. BARBU, J. PANIJELAND G. QUASH,Ann. Inst. Pasteur, IOO (1961) 725. 4 G. QUASH, J. P. DANDEU, E. BARBUAND J. PANIJEL, ~nn. Inst. Pasteur, lO3 (1962) 3. i A. TISSI~RESAND G. WATSON,Nature, 182 (1958) 778. ¢ S. DAGLEYAND J. SYKES, Biochem. J., 74 (196o) liP. '1 R. J. BRITTEN, B. J. MCCARTHYAND R. B. ROBERTS,Biophys. J., 2 (1962) 83. s A. TlSSI~RES, O. SCHLESSINGERAND F. GROS, Pr0c. Natl. Acad. Sci. U.S., 46 (196o) 145o. o H. LAMFROMAND E. GLOWACKI,J. Mol. Biol., 5 (1962) 97. Received, December 24th, 1962 Biochim. Biophys. Acta, 68 (I963) 330-332
P R E L I M I N A R Y NOTES
I F. H. WILT AND T. I-tULTIN, Biochem. Biophys. Res. Commun., 9 (1962) 313 . * A, C. GRIFFINANDM. A. O'NEAL, Biochim. Biophys. Acta, 61 (1962) 469 . * H. I~..V. ARNSTEIN, R. A. COX AND J. A. HUNT, Nature, 194 (1962) lO42. 4 j. G. FLAKS, E. C. COX AND J. R. \VHITE, Biochem. Biophys. Res. Commun., 7 (1962) 385. $ S. H. BARONDESAND M. W. NIRENBERG,Science, 138 (1962) 81o. Received J a n u a r y 3rd, 1963 Biochim. Biophys..4cta, 68 (1963) 328-33o
PN 6086 The distribution of protein antigens among Escherichia coli ribosomes In previous communications 1-4 we have presented qualitative as well as quantitative evidence defining the antigenic community existing between ribosomes of various species at the level of R N A for "distant heterologous ribosomes", and at the RNA and protein level for "neighbouring heterologous ribosomes". Homologous ribosomes possess in addition to the two aforementioned types, specific antigens of a protein nature. In order to assess whether these ribosomal protein antigens were present on all ribosomes or restricted to certain, goat-serum anti-ribosomes of Escherichia coli KI2 3000 (obtained b y twice weekly intravenous inlections, each of 6 mg of ribosomes) was therefore fractionated into its component antibodies by complete successive absorptions with the ribosomes of Welchia per/ringens, Alcaligenes /aecalis, Proteus vulgaris, E. coli K12 3000 Del X74 and E. coli K~2 3300. To the antibodies recuperated (as described below, from the specific precipitates formed successively with the ribosomes of Welchia per/ringens, Alcaligenes /aecalis, and Proteus vulgaris, were then added the ribosomes of E. coli K12 3000. In another series of experiments, E. coli ribosomes were added to the antibodies remaining in the supernatant after complete absorption by the ribosomes of Proteus vulgaris, E. coli K~2 3000 Del X74 and E. coli K12 3300. These precipitation reactions were carried out as previously described s. The RNA content of the precipitate ribosomes was further verified by concomitant estimation of the RIgA remaining in the supernatant. Ribosomes prepared essentially by the technique of TISSIIkRES AND WATSON 5, were purified b y 4 successive cycles of centrifugation in the Spinco preparative centrifuge, and finally suspended in o.oi M magnesium acetate, 0.005 M Tris-HC1 buffer (pH 7-4). The specific precipitates formed on addition of ribosomes to the antiserum were twice washed with a solution containing o.oi M magnesium acetate, o.14 M NaC1, 0.005 M Tris-HC1 buffer (pH 7-5) and then incubated at 37 ° for 18 h in o.14 M NaC1, 0.005 M Tris-HC1 buffer (pH 8.5). After centrifuging the resulting suspension, the antibodies were precipi'tated from the supernatant b y (N'H4)2SO4 to 0.35 saturation at 4 ° and finally solubilised in o.14 M NaC1 (pH 7.5)After each complete absorption of the antiserum b y the ribosomes of the different species indicated above, the excess ribosomes remaining in the supernatan~ were eliminated b y centrifuging the serum in the Spinco preparative centrifuge at IOOOOOXg for 4 h. As shown in Fig. I, with the "antibody fraction Welchia per[ringens", the "antibody fraction Alcaligenes/aecalis" and the "antibody fraction Proteus vulgaris", Biochim. Biophys. Acta. 68 (1963) 330-332
331
PRELIMINARY NOTES "Antibody
100,
8C 9
,
fraction" :
0 = Welchio perfringens X = AIcQligenes foecalis Z~ = P r o t e u s v u l g o r i s
6O
a. 4 0 < ~
20
400
1200 2000 2800 3600 4400 ~ug RNA added to l a g of antibody
Fig.
Serum
antidbosomes Of Kla. 3 0 0 0 absorbed byribosomes of:
40
9 ~8
I
E.Coli
0 = Proteus vulgoris iX =E, ColI K12 3 0 0 0 DelX74 FI =E.Coll Kt2 3 3 0 0
3G
~. 2C < z t~ 1C
200
I
I
I
600 1000 1400 1800 2200 ~Jg RNA added to 1mr of anti-serum Fig.
2
(antibodies recuperated from the corresponding specific precipitates as outlined above) the percentage of RNA precipita ted attains or tends to IOO % under conditions of excess antibody. TO determine whether these three types of antibodies were directed against RNA or protein, similar precipitation reactions were carried out with the ribosomes of Fusi/ormis/usi/ormis a "distant heterologue ''z. The percentage of RNA precipitated was IOO ~o with the "antibody fraction Welchia per/ringens", whilst no precipitation was obtained with the "antibody fraction Alcaligenes /aecalis" and the "antibody fraction Proteus vulgaris". Thus, the latter two antibody fractions are exclusively directed against protein antigens. The lower percentages (Fig. 2) obtained with the antibodies remaining in the supernatant after complete absorption of the goat antiserum by the ribosomes of Proteus vulgaris (A), E. eoli K12 3000 Del X74(B ) and E. coli K12 3300 (C) could not be ascribed to the existence of soluble complexes, since the repetition of these experiments with the addition of rabbit-serum anti-goat v-globulin to the supernatant gave no further precipitation of RN'A. That these antigens (identified as basic proteins in immunoelectrophoresis4) are composite parts of E. coli ribosomes and not independent ribonucleoprotein particles can be deduced from the fact that, while the antibodies corresponding to those antigens are not absorbed by the ribosomes of Proteus vulgaris, yet the "antibody fraction Proteus vulgaris" directed against other ribosomal proteins precipitates IOO % of the ribosomes estimated by means of the Biochim. Biophys, Acta,
68 ( I 9 6 3 ) 3 3 0 - 3 3 2
332
PRELIMINARY NOTES
RNA. Neither does the purification procedure eliminate measurable quantities of these antigens from the ribosomes, since identical precipitation curves are obtained with ribosomes prepared in dissociating medium: o.ooi M magnesium acetate--o.o5 M Tris-HC1 buffer (pH 7.5). We can therefore conclude, t h a t these lower percentages of ribosomal R N A in the precipitates obtained with the antisera (A) (B) and (C) indicate the existence of heterogeneity a m o n g the component particles of a ribosome preparation. An a t t e m p t was therefore m a d e to determine at what stage during the formation of ribosomes these antigens appeared. To an exponential culture of E. coli was added IOO /zg/ml of chloramphenicol and the cells were harvested after I h. The ribosomes from these cells gave with antiserum (C) a slight increase of about 15 % of antibodies as well as R N A precipitated, as compared to ribosomes from untreated cells. Since the ribonucleoprotein accumulating in the presence of chloramphenicol eventually becomes 3o-S particles e,7 this result indicates that the antigens reacting with antiserum (C) are found on at least the 3o-S subunits, but p r o b a b l y not with all, since the percentage increase of the R N A precipitated is relatively small. The main feature of this investigation lies in the heterogenous distribution of the antigens linked to the characteristics of the strain, compared to the homogenous distribution found for the ribosomal common antigens. We are therefore investigating whether the ribonucleoprotein precipitated b y antiserum (C) represents specialised ribosomes in view of the fact t h a t TISSI~RES et al. s with E. coli ribosomes, and LAMFROM AND GLOWACKI9 with the ribosomes of rabbit reticulocytes have found that a similar fraction (5-1o %) of the ribosomes of a population which are resistant to dissociation possess increased incorporating ability. The a u t h o r gratefully acknowledges the kind encouragement and valuable discussions of Dr. E. BARBLr t h r o u g h o u t this investigation. Service du Dr. Wahl,
GERARD QUASH
Institut Pasteur (Paris)
x E. BARBI~,J. PANIJEL, P. CAYEUXAND R. WAHL, Compt. Rend., 249 (1959) 338. I •. BARBU AND J. PANIJEL, Compt. Rend., 25° (196o) 1382. * E. BARBU, J. PANIJELAND G. QUASH,Ann. Inst. Pasteur, IOO (1961) 725. 4 G. QUASH, J. P. DANDEU, E. BARBUAND J. PANIJEL, ~nn. Inst. Pasteur, lO3 (1962) 3. i A. TISSI~RESAND G. WATSON,Nature, 182 (1958) 778. ¢ S. DAGLEYAND J. SYKES, Biochem. J., 74 (196o) liP. '1 R. J. BRITTEN, B. J. MCCARTHYAND R. B. ROBERTS,Biophys. J., 2 (1962) 83. s A. TlSSI~RES, O. SCHLESSINGERAND F. GROS, Pr0c. Natl. Acad. Sci. U.S., 46 (196o) 145o. o H. LAMFROMAND E. GLOWACKI,J. Mol. Biol., 5 (1962) 97. Received, December 24th, 1962 Biochim. Biophys. Acta, 68 (I963) 330-332