I M M U N O C H E M I C A L ASSAY F O R I N F O R M A T I N IN C Y T O P L A S M I C POLYSOMES
E. M. L U K A N I D I N
Institute of Molecular Biology, Academy of Sciences of the U.S.S.R., Moscow B-312, U.S.S.R. and F. N O L L
Central Institute of Molecular Biology, Department of Cell Physiology, Academy of Sciences of GDR, 1115 Berlin-Buch, G.D.R.
(Received 15 March, 1977 at Moscow) (Received 6 May, 1977 at Nijmegen) Abstract. Only very small amounts of informatin could be detected in cytoplasmic polysomes with the aid of antiinformatin antibodies. This low amount of informatin cannot represent a structural component of polysomes or polysomal mRNP.
I.
INTRODUCTION
It is known that practically all pre-mRNA in the nuclei of mammalian cells is bound with specific protein particles called 'informofers' [ 1-3]. One informofer contains a number of homogeneous protein molecules designated as informatin. One or two types of polypeptides were found in informatin [4]. In the cytoplasm mRNA is present in the form of polysomal mRNA-protein complexes [5-6] and as free'informosomes' [7]. The polypeptides of polysomal m R N P particles and those of informatin behave differently when eleetrophoresed in urea gels [8]. This was now further analysed with the aid of immunological methods which seem to be more sensitive and therefore may allow to see whether polypeptides of the informofers are involved in the formation of polysomal mRNP~ Here we describe experiments using anti-informatin antibodies as a specific tool for detecting informatin in rat liver polysomes. From the results we conclude that only a very small amount ofinformatin could be detected in polysomes. II.
MATERIAL AND METHODS
The methods for obtaining nuclei and isolation of nuclear RNP particles, have been previously described [2]. Informatin was prepared from purified 30S particles which were 347 Molecular Biology Reports 3 (1977) 347-351, A II Rights Reserved. Copyright 9 1977 by D. Reidel Publishing Company, Dordrecht-Holland.
concentrateci by ammonium sulfate precipitation, After centrifugation the pellet of 30S RNP was resuspended in 2 M NaCI and dialysed against 2 M NaC1 overnight. Precipitated RNA was removed by centrifugation (30 min at 15000• at 0 ~C). Antisera against 30S particles and against informatin of rat liver were obtained by immunization of rabbits or hens as has been described [9]. The antigen-antibody complexes were analyzed by the methods of passive hemagglutination [ 10] and inhibition of immunoadsorption [I 1]. The immunoadsorbent was prepared according to Rokhlin and Nezlin [ 11] by fixation of immunoglobulins from anti-informatin antiserum on cellulose, lmmunoadsorbent with immunoglobulins from non-immune serum was prepared as a control. At first the unlabeled antige~a (polysomes or 30S particles) in I ml of 10% inactivated calf serum in saline (CSS) was added to 0.1 ml of antibody-immunoadsorbents (containing 100 ~g immunoglobulins). After 30 min incubation at room temperature with shaking the [125I] _ labeled informatin in 2 ml CSS was added and the mixture was incubated for a further 60 min. Then the immunoadsorbents were washed with CSS 5 times to remove nonbound labeled informatin. The radioactivity bound to the immunoadsorbents was measured in a gamma-counter ('USS-I' U.S.S.R.). Immunoadsorption of [125I]-informatin in the absence of unlabeled antigens (100% of binding) was of about 4500 cpm. Iodination of informatin was performed according to Bale et al. [ 12]. No more than 1% of free 125Iwas present in the preparation of[~25I]-informatin; the specific activity was 3 • 105 cpm/~g. Three techniques for polysome isolation were used: (1) Free polysomes were prepared according to Blobel and Potter [ 13] without detergents using cushions of 1.3 M and 2.0 M sucrose containing 0.025 M Tris-HCl, pH 7.6 - 0.05 M KC1 - 0.005 M MgC12 for purification of polysomes (Spinco Ti50 rotor, 4 hours at 46 000 rpm at 0 ~ C). The pellet was resuspended in a buffer of the same ionic composition. During the last step precautions were taken to avoid contamination with other layers. (2) Polysomes were prepared by the method of Olsnes [ 14] with 2% Triton-X- 100. The preparations were sedimented through a cushion of 2.0 M sucrose containing 0.01 M Triethanolamine-HCl, pH 7.4 - 0.15 M KCI - 0.001 M MgC12. The pellet was treated as described above for free polysomes. (3) Polysomes were prepared by the same procedure of Olsnes but instead of Triton X-100, 1% Na-deoxycholate was used: Ribosomes from rat liver and total ribosomal protein were prepared as described earlier [15-16]. III.
RESULTS AND DISCUSSION
It was shown previously that protein of polysomal m R N P are not identical with those of nuclear 30S particles as evidenced from the difference in their mobility in polyacrylamide gel electrophoresis [8]. Moreover, it could be demonstrated by a special immunological technique using antibodies against 30S particles that these particles are not associated with cytoplasmic polysomes [9]. In the experiments presented in this report the reactivity of polysomes was studied in 348
hemagglutination tests with antisera against informatin and 30S RNP particles. Three kinds of polysomes from rat liver were prepared to check the influence of detergents and ionic strength on their immunological reactivity. TABLE I: Hemagglutination titres ofantisera against 30S particles, informatin and total ribosomal protein with different kinds of polysomes and informatin as control antigen
Antigen tested
Rabbit antiserum against 30S particles
informatin
Hen antiserum against 30S particles
Rabbit antiserum against ribosomal
Free polysomes Polysomes (0.15 KC1 and Triton X- 100)
0
1:640
0
1:20 480
0
1:160
0
1:20 480
Polysomes (0.15 KCI and DOC)
0
0
0
1:10 240
80S ribosomes
0
0
0
1:20 480
Ribosomal protein
0
0
0
1:10 240
Informatin
1:5 !20
1:20 480
1:640
0
Table I shows that antisera against 30S particles, prepared from rabbit and hen, did not react with polysomes prepared in any of three ways described above. Thus 30S nuclear RNP particles as structural units are absent in cytoplasmic polysomes. This is in agreement with earlier findings [9]. On the other hand, serum against informatin gave a positive immunereaction with free polysomes, and to a lower extent, with polysomes, prepared by Triton treatment in high KC1 concentrations. Na-deoxycholate treated polysomes did not react at all. As could be expected, the titers of hemagglutination with polysomes were much lower as compared to informatin. 80S ribosomes as well as isolated total ribosomal protein were inactive with both types of antiserum. As a control, antiserum against total ribosomal protein reacted with all kinds of polysomes, but not with informatin. Therefore, one can suggest that informatin, though in small amounts, is present in cytoplasmic polysomes as a constituent of the mRNA-protein complex. Now the question arises as to whether the inforrnatin found in polysomes by antiinformatin antibodies is due to contaminating material from the nucleus, or whether it is a natural component of the polysomes. The fact that even polysomes prepared under conditions preventing contamination by nuclear material [ 14] showed a positive immunereaction with anti-informatin antibodies, indicates that informatin is not a simple contami349
nation of polysomes. Polysomes treated with Na-deoxycholate lose their reactivity with antiinformatin antibodies. Thus, treatment ofpolysomes with Na-deoxycholate causes removal of protein as well as informatin from polysomes. It is unlikely that informatin in polysomes represents nascent polypeptide chains of informatin because the latter have a very long half-life.
100
r
/
O !
0,,I NI--
.//'-"
10
0 o 0
c-
*
*
,
10
20
30
/I
0
I!
1200
RNP added (}Jg)
Fig. 1. Inhibitionof immunoadsorptionof [~25I]-labeledinformatin on anti-informatinimmunoadsorbentby different amounts of po lysomes (O--O) and 30S particles (*--*) as the cytoplasmic and nuclear RNP, respectively.
In order to estimate quantitatively the amount of informatin in polysomes we applied the method of inhibition of binding of [~2sI]-labeled informatin to an anti-informatin immunoadsorbent by polysomes. Figure I illustrates the results. Obviously 30S particles inhibit the binding of[nsI]-informatin very strongly; as little as 1 ~g causes about a 10% inhibition. In contrast, polysomes have no significant inhibitory effect even in amounts as great as 1200 #g. This means that 1200 ~g polysomes contain less than 1 ~g informatin. The protein content in polysomes amounts to about 50% [ 17]. Thus less than 0.16% of total polysomal protein may represent informatin. Taking into account a molecular weight of informatin equal to 40 000 one can calculate that in polysomes less than 0.1 molecule of informatin per ribosome is present. Each ribosome is bound to a piece of mRNA of approximately 3• liP daltons that corresponds to about lX 105 dalton of the polysomal mRNA-protein complex. Therefore less than 4% of the protein moiety of the polysomal m R N P corresponds to informatin. From this fact we may conclude that informatin cannot represent.a structural component of polysomal mRNP. The significance of this very small amount ofinformatin which was found in cytoplasmic polysomes is not clear. In our previous paper [ 18] we have postulated that informofer transports mRNA from chromatin to the nuclear membrane, whereas in the cytoplasm 350
mRNA is bound to another, cytoplasmic protein forming'free informosomes' or polysomal mRNP. The data presented in this paper are in agreement with the idea that the informofer per se never crosses the nuclear membrane. ACKNOWLEDGEMENTS We thank Prof. G.P. Georgiev for his interest and encouragement and Dr. O.V. Rokhlin for the supply of amino cellulose and for his valuable advice concerning the immunoadsorption technique. REFERENCES 1. 2.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Samarina, O.P., Asriyan, l.S., and Georgiev, G.P., Dokl. Akad. Nauk U.S.S.R. 163, 1510(1965). Samarina, O.P., Lukanidin, E.M., M olnar, J., and Georgiev, G.P.,J. Mol. Bio133, 251 (1968). Lukanidin, E.M., Zalmanzon, E.S., Komaromi, L., Samarina, O.P., and Georgiev, G.P., Nature New Biol. 235, 193 (1972). Krichevskaya, A.A. and Georgiev, G.P., MoL Biol. (U.S.S.R.) 7, 168 (1973). Perry, R.P. and Kelley, D. E., J. Mol. Biol. 35, 37 (1968). Henshaw, E.C.,J. Mol. Biol. 36,401 (1968). Spirin, A.S., in Mechanisms of Protein Synthesis and its Regulation, r Bosch, North-Holland Publ., 1972. Lukanidin, E.M., Georgiev, G.P., and Williamson, R., FEBSLetters 19, 152 (1971). Lukanidin, E.M., Olsnes, S., and Pihl, A., Nature New BioL 2AO,90 (1972 ). Noll, F., and Bielka, H., Molec. Gen. Genetics 106, 106(1970). Rokhlin, O.V., Vengerova, T.I., and Nezlin, R.S., lmmunochemistry 8, 525 (I 971). Bale, W.F., Helmkamp, R.W., Davis, T.P., Izzo, M.I., Goodland, R.L., Contreras, M.A., and Spar, I.U, Proc. Soc. Exp. Biol. Med. Bull. 122,407 (1966). Blobel, E. and Potter, V.R., J. Mol. Biol. 26, 279 (1967). Olsnes, S., Biochirn. Biophys. A cta 213,149 (1970). NoU,F., Neumann, W., and Bielka, H., lmmunochemistry 10, 9 (1973). Fogel, S., and Sypherd, P.S., J. Bacteriol. 96, 358 (1968). Bielka, H., Welfle, H., B6ttger, M., and F6rster, W., Eur. J. Biochem. 9, 263 (1968). Samarina, O.P., Lukanidin, E.M., and Georgiev, G. P. in Protein Synthesis in Reproductive Tissue, 5th Karolinska Symposium on Research Methods in Reproductive Endocrinology, Geneva 1973.
35t
E. M. L U K A N I D I N
Institute of Molecular Biology, Academy of Sciences of the U.S.S.R., Moscow B-312, U.S.S.R. and F. N O L L
Central Institute of Molecular Biology, Department of Cell Physiology, Academy of Sciences of GDR, 1115 Berlin-Buch, G.D.R.
(Received 15 March, 1977 at Moscow) (Received 6 May, 1977 at Nijmegen) Abstract. Only very small amounts of informatin could be detected in cytoplasmic polysomes with the aid of antiinformatin antibodies. This low amount of informatin cannot represent a structural component of polysomes or polysomal mRNP.
I.
INTRODUCTION
It is known that practically all pre-mRNA in the nuclei of mammalian cells is bound with specific protein particles called 'informofers' [ 1-3]. One informofer contains a number of homogeneous protein molecules designated as informatin. One or two types of polypeptides were found in informatin [4]. In the cytoplasm mRNA is present in the form of polysomal mRNA-protein complexes [5-6] and as free'informosomes' [7]. The polypeptides of polysomal m R N P particles and those of informatin behave differently when eleetrophoresed in urea gels [8]. This was now further analysed with the aid of immunological methods which seem to be more sensitive and therefore may allow to see whether polypeptides of the informofers are involved in the formation of polysomal mRNP~ Here we describe experiments using anti-informatin antibodies as a specific tool for detecting informatin in rat liver polysomes. From the results we conclude that only a very small amount ofinformatin could be detected in polysomes. II.
MATERIAL AND METHODS
The methods for obtaining nuclei and isolation of nuclear RNP particles, have been previously described [2]. Informatin was prepared from purified 30S particles which were 347 Molecular Biology Reports 3 (1977) 347-351, A II Rights Reserved. Copyright 9 1977 by D. Reidel Publishing Company, Dordrecht-Holland.
concentrateci by ammonium sulfate precipitation, After centrifugation the pellet of 30S RNP was resuspended in 2 M NaCI and dialysed against 2 M NaC1 overnight. Precipitated RNA was removed by centrifugation (30 min at 15000• at 0 ~C). Antisera against 30S particles and against informatin of rat liver were obtained by immunization of rabbits or hens as has been described [9]. The antigen-antibody complexes were analyzed by the methods of passive hemagglutination [ 10] and inhibition of immunoadsorption [I 1]. The immunoadsorbent was prepared according to Rokhlin and Nezlin [ 11] by fixation of immunoglobulins from anti-informatin antiserum on cellulose, lmmunoadsorbent with immunoglobulins from non-immune serum was prepared as a control. At first the unlabeled antige~a (polysomes or 30S particles) in I ml of 10% inactivated calf serum in saline (CSS) was added to 0.1 ml of antibody-immunoadsorbents (containing 100 ~g immunoglobulins). After 30 min incubation at room temperature with shaking the [125I] _ labeled informatin in 2 ml CSS was added and the mixture was incubated for a further 60 min. Then the immunoadsorbents were washed with CSS 5 times to remove nonbound labeled informatin. The radioactivity bound to the immunoadsorbents was measured in a gamma-counter ('USS-I' U.S.S.R.). Immunoadsorption of [125I]-informatin in the absence of unlabeled antigens (100% of binding) was of about 4500 cpm. Iodination of informatin was performed according to Bale et al. [ 12]. No more than 1% of free 125Iwas present in the preparation of[~25I]-informatin; the specific activity was 3 • 105 cpm/~g. Three techniques for polysome isolation were used: (1) Free polysomes were prepared according to Blobel and Potter [ 13] without detergents using cushions of 1.3 M and 2.0 M sucrose containing 0.025 M Tris-HCl, pH 7.6 - 0.05 M KC1 - 0.005 M MgC12 for purification of polysomes (Spinco Ti50 rotor, 4 hours at 46 000 rpm at 0 ~ C). The pellet was resuspended in a buffer of the same ionic composition. During the last step precautions were taken to avoid contamination with other layers. (2) Polysomes were prepared by the method of Olsnes [ 14] with 2% Triton-X- 100. The preparations were sedimented through a cushion of 2.0 M sucrose containing 0.01 M Triethanolamine-HCl, pH 7.4 - 0.15 M KCI - 0.001 M MgC12. The pellet was treated as described above for free polysomes. (3) Polysomes were prepared by the same procedure of Olsnes but instead of Triton X-100, 1% Na-deoxycholate was used: Ribosomes from rat liver and total ribosomal protein were prepared as described earlier [15-16]. III.
RESULTS AND DISCUSSION
It was shown previously that protein of polysomal m R N P are not identical with those of nuclear 30S particles as evidenced from the difference in their mobility in polyacrylamide gel electrophoresis [8]. Moreover, it could be demonstrated by a special immunological technique using antibodies against 30S particles that these particles are not associated with cytoplasmic polysomes [9]. In the experiments presented in this report the reactivity of polysomes was studied in 348
hemagglutination tests with antisera against informatin and 30S RNP particles. Three kinds of polysomes from rat liver were prepared to check the influence of detergents and ionic strength on their immunological reactivity. TABLE I: Hemagglutination titres ofantisera against 30S particles, informatin and total ribosomal protein with different kinds of polysomes and informatin as control antigen
Antigen tested
Rabbit antiserum against 30S particles
informatin
Hen antiserum against 30S particles
Rabbit antiserum against ribosomal
Free polysomes Polysomes (0.15 KC1 and Triton X- 100)
0
1:640
0
1:20 480
0
1:160
0
1:20 480
Polysomes (0.15 KCI and DOC)
0
0
0
1:10 240
80S ribosomes
0
0
0
1:20 480
Ribosomal protein
0
0
0
1:10 240
Informatin
1:5 !20
1:20 480
1:640
0
Table I shows that antisera against 30S particles, prepared from rabbit and hen, did not react with polysomes prepared in any of three ways described above. Thus 30S nuclear RNP particles as structural units are absent in cytoplasmic polysomes. This is in agreement with earlier findings [9]. On the other hand, serum against informatin gave a positive immunereaction with free polysomes, and to a lower extent, with polysomes, prepared by Triton treatment in high KC1 concentrations. Na-deoxycholate treated polysomes did not react at all. As could be expected, the titers of hemagglutination with polysomes were much lower as compared to informatin. 80S ribosomes as well as isolated total ribosomal protein were inactive with both types of antiserum. As a control, antiserum against total ribosomal protein reacted with all kinds of polysomes, but not with informatin. Therefore, one can suggest that informatin, though in small amounts, is present in cytoplasmic polysomes as a constituent of the mRNA-protein complex. Now the question arises as to whether the inforrnatin found in polysomes by antiinformatin antibodies is due to contaminating material from the nucleus, or whether it is a natural component of the polysomes. The fact that even polysomes prepared under conditions preventing contamination by nuclear material [ 14] showed a positive immunereaction with anti-informatin antibodies, indicates that informatin is not a simple contami349
nation of polysomes. Polysomes treated with Na-deoxycholate lose their reactivity with antiinformatin antibodies. Thus, treatment ofpolysomes with Na-deoxycholate causes removal of protein as well as informatin from polysomes. It is unlikely that informatin in polysomes represents nascent polypeptide chains of informatin because the latter have a very long half-life.
100
r
/
O !
0,,I NI--
.//'-"
10
0 o 0
c-
*
*
,
10
20
30
/I
0
I!
1200
RNP added (}Jg)
Fig. 1. Inhibitionof immunoadsorptionof [~25I]-labeledinformatin on anti-informatinimmunoadsorbentby different amounts of po lysomes (O--O) and 30S particles (*--*) as the cytoplasmic and nuclear RNP, respectively.
In order to estimate quantitatively the amount of informatin in polysomes we applied the method of inhibition of binding of [~2sI]-labeled informatin to an anti-informatin immunoadsorbent by polysomes. Figure I illustrates the results. Obviously 30S particles inhibit the binding of[nsI]-informatin very strongly; as little as 1 ~g causes about a 10% inhibition. In contrast, polysomes have no significant inhibitory effect even in amounts as great as 1200 #g. This means that 1200 ~g polysomes contain less than 1 ~g informatin. The protein content in polysomes amounts to about 50% [ 17]. Thus less than 0.16% of total polysomal protein may represent informatin. Taking into account a molecular weight of informatin equal to 40 000 one can calculate that in polysomes less than 0.1 molecule of informatin per ribosome is present. Each ribosome is bound to a piece of mRNA of approximately 3• liP daltons that corresponds to about lX 105 dalton of the polysomal mRNA-protein complex. Therefore less than 4% of the protein moiety of the polysomal m R N P corresponds to informatin. From this fact we may conclude that informatin cannot represent.a structural component of polysomal mRNP. The significance of this very small amount ofinformatin which was found in cytoplasmic polysomes is not clear. In our previous paper [ 18] we have postulated that informofer transports mRNA from chromatin to the nuclear membrane, whereas in the cytoplasm 350
mRNA is bound to another, cytoplasmic protein forming'free informosomes' or polysomal mRNP. The data presented in this paper are in agreement with the idea that the informofer per se never crosses the nuclear membrane. ACKNOWLEDGEMENTS We thank Prof. G.P. Georgiev for his interest and encouragement and Dr. O.V. Rokhlin for the supply of amino cellulose and for his valuable advice concerning the immunoadsorption technique. REFERENCES 1. 2.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Samarina, O.P., Asriyan, l.S., and Georgiev, G.P., Dokl. Akad. Nauk U.S.S.R. 163, 1510(1965). Samarina, O.P., Lukanidin, E.M., M olnar, J., and Georgiev, G.P.,J. Mol. Bio133, 251 (1968). Lukanidin, E.M., Zalmanzon, E.S., Komaromi, L., Samarina, O.P., and Georgiev, G.P., Nature New Biol. 235, 193 (1972). Krichevskaya, A.A. and Georgiev, G.P., MoL Biol. (U.S.S.R.) 7, 168 (1973). Perry, R.P. and Kelley, D. E., J. Mol. Biol. 35, 37 (1968). Henshaw, E.C.,J. Mol. Biol. 36,401 (1968). Spirin, A.S., in Mechanisms of Protein Synthesis and its Regulation, r Bosch, North-Holland Publ., 1972. Lukanidin, E.M., Georgiev, G.P., and Williamson, R., FEBSLetters 19, 152 (1971). Lukanidin, E.M., Olsnes, S., and Pihl, A., Nature New BioL 2AO,90 (1972 ). Noll, F., and Bielka, H., Molec. Gen. Genetics 106, 106(1970). Rokhlin, O.V., Vengerova, T.I., and Nezlin, R.S., lmmunochemistry 8, 525 (I 971). Bale, W.F., Helmkamp, R.W., Davis, T.P., Izzo, M.I., Goodland, R.L., Contreras, M.A., and Spar, I.U, Proc. Soc. Exp. Biol. Med. Bull. 122,407 (1966). Blobel, E. and Potter, V.R., J. Mol. Biol. 26, 279 (1967). Olsnes, S., Biochirn. Biophys. A cta 213,149 (1970). NoU,F., Neumann, W., and Bielka, H., lmmunochemistry 10, 9 (1973). Fogel, S., and Sypherd, P.S., J. Bacteriol. 96, 358 (1968). Bielka, H., Welfle, H., B6ttger, M., and F6rster, W., Eur. J. Biochem. 9, 263 (1968). Samarina, O.P., Lukanidin, E.M., and Georgiev, G. P. in Protein Synthesis in Reproductive Tissue, 5th Karolinska Symposium on Research Methods in Reproductive Endocrinology, Geneva 1973.
35t
