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Effect of the S-100 protein on the RNA-polymerase activity in brain nucleoli isolated from newborn rat
Neuroscience Letters, 2 (1976) 23--27 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
23
E F F E C T OF THE S-100 PROTEIN ON THE RNA-POLYMERASE ACTIVITY IN B R A I N NUCLEOLI ISOLATED F R O M NEWBORN RAT*
FABRIZIO MICHETTI and GABRIELLA DeRENZIS Instituto di Anatomia Umana, Universit~ Cattolica, 00168 Roma (Italy) (Received January 9th, 1976) (Accepted January 16th, 1976)
SUMMARY
The structural integrity of the nucleus is not a prerequisite for the action of the S-100 protein on the RNA-polymerase activity in brain nuclei. In fact the protein also stimulates the enzyme activity in sonicated nuclei of newborn rat, as well as directly in isolated nucleoli from the same source.
The brain-specific S-100 protein [ 14 ] is present b o t h in glial cells [ 1,4 ] and neurons [15,17] in a soluble [14] as well as in a membrane-bound form [5, 16]. S-100 has also been shown b y immunohistological procedures in the cell nucleus [8,18], where it has been measured in subnuclear fractions by immunochemical assays [12]. Previously, we have studied some properties of the nuclear S-100. Data have been obtained on the presence of the protein in brain chromatin and on its in vitro transfer into isolated nuclei [ 12]. A stimulating effect on the nucleolar RNA-polymerase activity in nuclei isolated from immature brain has also been demonstrated [11], and this action has been shown to be organ-specific to the brain (Michetti et al., submitted for publication). The present study is aimed at investigating whether the integrity of the nuclear structure is a prerequisite for the in vitro action of the protein or whether the S-100 affects directly the nucleolus. Data will be presented indicating that the p h e n o m e n o n is also elicited in isolated brain nucleoli. Nuclei from newborn rat brain were prepared according to the procedure described b y LCvtrup-Rein and McEwen [9] with minor modifications, as indicated elsewhere [12]. Nucleoli from the same source were isolated b y the sonication procedure [3]. The purity of the nucleolar preparation was verified b y light microscopic examination with toluidine blue staining and b y M/E examination (Fig. 1). The Mg:÷-dependent RNA-polymerase activity was determined both in sonicated nuclei and in isolated nucleoli according to the *A preliminary account of these data was presented at the Fifth International Meeting of the International Society for Neurochemistry, Barcelona, Spain, September 1975.
24
Fig. 1. Electron micrograph of brain nucleoli isolated from newborn rat. X 12,600. The nuclear pellet was resuspended in 2 ml of 0.25 M sucrose and sonicated with an MSE Ultrasonic Power Unit (1.5 A) until virtually all nuclei were broken (four times for 10 sec each time at 10 sec intervals). The sonicated suspension was diluted to 5 ml of the same solution, layered over 15 ml of 0.88 M sucrose and centrifuged at 2000 x g for 20 min. The pellet contained the purified nucleoli. All operations described were performed at 0--4°C. For M/E examination, the nucleolar pellet was fixed in glutaraldehyde 2% in phosphate buffer 100 raM, pH 7.3, postfixed in 1% osmium tetroxide in the same buffer, rapidly dehydrated in ethanol and embedded in Epon. p r o c e d u r e o f M o n t a n a r o et al. [ 1 3 ] , as indicated in Fig. 2. Nuclear as well as nucleolar D N A c o n c e n t r a t i o n s were m e a s u r e d b y t h e m e t h o d o f B u r t o n [ 2 ] . The S-100 p r o t e i n was p r e p a r e d a c c o r d i n g t o the p r o c e d u r e o f M o o r e [ 1 4 ] . a - A m a n i t i n was a g e n e r o u s gift o f Dr. DiMauro, University o f R o m e . Initially the stimulating a c t i o n o f t h e p r o t e i n was tested o n s o n i c a t e d nuclei (Fig. 2). In a c c o r d a n c e with t h e d a t a o f D u t t o n and Mahler [ 6 ] , t h e R N A - p o l y m e r a s e activity appears to be l o w e r in d i s r u p t e d nuclei t h a n in i n t a c t ones, b u t t h e S-100 also stimulates t h e e n z y m e activity u n d e r these c o n d i t i o n s . T h e R N A - p o l y m e r a s e activity was t h e n tested d i r e c t l y in isolated nucleoli in t h e presence or in t h e absence o f t h e S-100. Fig. 3 shows t h a t o u r nucleolar p r e p a r a t i o n s exhibit R N A s y n t h e t i c activity w h i c h is also s t i m u l a t e d b y t h e S-100 protein. As previously observed with isolated brain nuclei [ 1 1 ] , the stimulating effect o f S-100 was c o n f i r m e d in t h e presence o f a - a m a n i t i n .
25
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Fig. 2. Effect of the S-100 protein on the Mg~+-dependent RNA-polymerase activity in sonicated brain nuclei isolated from newborn rat. Sonicated nuclei (four times for 10 sec each time at 10 sec intervals at 1.5 A) were preincubated for 10 rain at 37°C in 100 mM tris-HC1 buffer pH 8.0 containing 4 mM MgC12, in the presence or in the absence of S-100 protein (200 ~g/ml). The assay was started in the reaction mixture at 37°C with 0.1 ml o f preincubated suspension, and was stopped after 15 rain with 5 ml of ice-cold perchloric acid (0.5 N) in 1% sodium pyrophosphate. One milligram of bovine serum albumin was added as a carrier. The precipitate was then washed twice more with 6 ml of cold perchloric acid (0.2 N) in 1% sodium pyrophosphate. The final precipitate was solubilized in 0.5 ml of NCS reagent (Nuclear Chicago Solubilizer) and 10 ml of toluene-based scintillation liquid were added for counting the radioactivity in a Nuclear Chicago scintillation spectrometer. Counting efficiency was approximately 35%. The activity was expressed in distint./min/mg DNA. D N A per assay tube: approximately 200 ug. The values are the average o f three experiments. Fig. 3. Time course of Mg2+-activated RNA-polymerase reaction in isolated brain nucleoli from newborn rat. The e n z y m e activity was determined as described in Fig. 2 for the indicated times. D N A per assay tube: 50 ~g. The values are the average o f four experiments.
This finding is obvious in isolated nucleoli, since ~-amanitin is a selective inhibitor of the nucleoplasmic RNA-polymerase activity. On the contrary, no stimulating activity was observed in the presence of bovine serum albumin, poly-/-aspartate (M.W. 4870) or poly-l-glutamate (M.W. 19,700) employed as a control {not shown). The above data give additional information about the properties of the
I
26 nuclear f r a c t i o n o f S-100, b u t at present it is impossible t o state to w h a t ext e n t these in vitro findings m a y be c o r r e l a t e d to t h e in vivo behavior o f the protein. With these limitations in mind, the f o l l o w i n g c o n s i d e r a t i o n s m a y be m a d e : (1) the integrity of t h e nuclear s t r u c t u r e is n o t a prerequisite for the a c t i o n o f the p r o t e i n ; (2) the S-100 p r o t e i n does n o t e x e r t its s t i m u l a t o r y activity m e r e l y b y e n h a n c i n g the e n t r a n c e into the nucleus o f R N A precursors; (3) the S-100 p r o t e i n is able to stimulate directly n a k e d nucleoli, alt h o u g h t h e possibility for c o o p e r a t i v e actions b y n u c l e o p l a s m i c a n d / o r nuclear m e m b r a n o u s c o m p o n e n t s in i n t a c t nuclei c a n n o t be ruled out. Since nucleoli are believed t o lack tissue specificity [ 1 9 ] , it is intriguing t h a t t h e y are s t i m u l a t e d b y a brain-specific protein, w h i c h f u r t h e r m o r e affects specifically brain nuclei. ACKNOWLEDGEMENTS T h e a u t h o r s are m u c h i n d e b t e d to Prof. N. Miani for critical review o f the m a n u s c r i p t , and t o Dr. C. Olivieri-Sangiacomo f o r M/E e x a m i n a t i o n s o f nucleolar preparations. This investigation was partially s u p p o r t e d b y C.N.R. C o n t r a c t No. 74.00229.04. REFERENCES 1 Benda, P., Lightbody, J., Sato, G., Levine, L., and Sweet, W.A., Differentiated rat glial cell strain in tissue culture, Science, 161 (1968) 370--371. 2 Burton, K., A study or" the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid, Biochem. J., 62 (1956) 315--323. 3 Busch, H., Isolation and purification of nucleoli. In L. Grossman and K. Moldave (Eds.), Methods in Enzymology, Volume XII, Part A, Academic Press, New York, 1967, pp. 448--464. 4 Cicero, T.J., Cowan, W.M., Moore, B.W., and Suntzeff, V., The cellular localization of the two brain specific proteins S-100 and 14.3.2, Brain Res., 18 (1970) 25--34. 5 Donato, R., Michetti, F., and Miani, N., Soluble and membrane-bound S-100 protein in cerebral cortex synaptosomes. Properties of the S-100 receptor, Brain Res., 98 (1975) 561--573. 6 Dutton, G.H., and Mahler, H.R., In vitro RNA synthesis by intact rat brain nuclei, J. Neurochem., 15 (1968) 765--780. 7 Haglid, K., and Stavrou, D., Water-soluble and pentanol-extractable proteins in human brain tissue and human brain tumours, with special reference to S-100 protein, J. Neurochem., 20 (1973) 1523--1532. 8 Hyd~n, H., and McEwen, B.S., A glial protein specific for the nervous system, Proc. nat. Acad. Sci. (Wash.), 55 (1966) 354--358. 9 L~bvtrup-Rein, H., and McEwen, B.S., Isolation and fractionation of rat brain nuclei, J. Cell Biol., 30 (1966) 405--415. 10 Miani, N., DeRenzis, G., Michetti, F., Correr, S., Olivieri-Sangiacomo, C., and Caniglia, A., Axonal transport of S-100 protein in mammalian nerve fibres, J. Neurochem., 19 (1972) 1387--1394. 11 Miani, N., Michetti, F., DeRenzis, G., and Caniglia, A., Effect of a brain specific protein
27
12 13
14 15 16 17 18 19
(S-100 protein) on the nucleolar RNA-polymerase activity in isolated brain nuclei, Experientia (Basel), 29 (1973) 1499--1501. Michetti, F., Miani, N., DeRenzis, G., Caniglia, A., and Correr, S., Nuclear localization of S-100 protein, J. Neurochem., 22 (1974) 239--244. Montanaro, N., Novello, F., and Stirpe, F., Effect of ~-amanitin on ribonucleic acid polymerase II of rat brain nuclei and on retention of avoidance conditioning, Biochem. J., 125 (1971) 1087--1090. Moore, B.W., A soluble protein characteristic of the nervous system, Biochem. biophys. Res. Commun., 19 (1965) 739--744. Packman, P.M., Blomstrand, C., and Hamberger, A., Disc electrophoretic separation of proteins in neuronal, glial, and subcellular fractions from cerebral cortex, J. Neurochem., 18 (1971) 479--487. Rusca, G., Calissano, P., and Alem~, S., Identification of a membrane-bound fraction of the S-100 protein, Brain. Res., 49 (1972) 223--227. Schubert, D., Heinemann, S., Carlish, W., Tarikas, H., Kimes, B., Patrick, J., Steinbach, J.H., Culy, W., and Brandt, B.L., Clonal cell lines from rat central nervous system, Nature (Lond.), 249 (1974) 224--227. Sviridov, S.M., Korochin, L.I., Ivanov, V.N., Maletskaya, E.I., and Bakhtina, T.K., Immunohistochemical studies of S-100 protein during post-natal ontogenesis of the brain of two rat strains, J. Neurochem., 19 (1972) 713--718. Takahashi, Y., Araki, K., Ikeda, K., and Oyanagi, S., Isolation and some characteristics of nucleoli from the brain, Brain Res., 73 (1974) 189--203.
23
E F F E C T OF THE S-100 PROTEIN ON THE RNA-POLYMERASE ACTIVITY IN B R A I N NUCLEOLI ISOLATED F R O M NEWBORN RAT*
FABRIZIO MICHETTI and GABRIELLA DeRENZIS Instituto di Anatomia Umana, Universit~ Cattolica, 00168 Roma (Italy) (Received January 9th, 1976) (Accepted January 16th, 1976)
SUMMARY
The structural integrity of the nucleus is not a prerequisite for the action of the S-100 protein on the RNA-polymerase activity in brain nuclei. In fact the protein also stimulates the enzyme activity in sonicated nuclei of newborn rat, as well as directly in isolated nucleoli from the same source.
The brain-specific S-100 protein [ 14 ] is present b o t h in glial cells [ 1,4 ] and neurons [15,17] in a soluble [14] as well as in a membrane-bound form [5, 16]. S-100 has also been shown b y immunohistological procedures in the cell nucleus [8,18], where it has been measured in subnuclear fractions by immunochemical assays [12]. Previously, we have studied some properties of the nuclear S-100. Data have been obtained on the presence of the protein in brain chromatin and on its in vitro transfer into isolated nuclei [ 12]. A stimulating effect on the nucleolar RNA-polymerase activity in nuclei isolated from immature brain has also been demonstrated [11], and this action has been shown to be organ-specific to the brain (Michetti et al., submitted for publication). The present study is aimed at investigating whether the integrity of the nuclear structure is a prerequisite for the in vitro action of the protein or whether the S-100 affects directly the nucleolus. Data will be presented indicating that the p h e n o m e n o n is also elicited in isolated brain nucleoli. Nuclei from newborn rat brain were prepared according to the procedure described b y LCvtrup-Rein and McEwen [9] with minor modifications, as indicated elsewhere [12]. Nucleoli from the same source were isolated b y the sonication procedure [3]. The purity of the nucleolar preparation was verified b y light microscopic examination with toluidine blue staining and b y M/E examination (Fig. 1). The Mg:÷-dependent RNA-polymerase activity was determined both in sonicated nuclei and in isolated nucleoli according to the *A preliminary account of these data was presented at the Fifth International Meeting of the International Society for Neurochemistry, Barcelona, Spain, September 1975.
24
Fig. 1. Electron micrograph of brain nucleoli isolated from newborn rat. X 12,600. The nuclear pellet was resuspended in 2 ml of 0.25 M sucrose and sonicated with an MSE Ultrasonic Power Unit (1.5 A) until virtually all nuclei were broken (four times for 10 sec each time at 10 sec intervals). The sonicated suspension was diluted to 5 ml of the same solution, layered over 15 ml of 0.88 M sucrose and centrifuged at 2000 x g for 20 min. The pellet contained the purified nucleoli. All operations described were performed at 0--4°C. For M/E examination, the nucleolar pellet was fixed in glutaraldehyde 2% in phosphate buffer 100 raM, pH 7.3, postfixed in 1% osmium tetroxide in the same buffer, rapidly dehydrated in ethanol and embedded in Epon. p r o c e d u r e o f M o n t a n a r o et al. [ 1 3 ] , as indicated in Fig. 2. Nuclear as well as nucleolar D N A c o n c e n t r a t i o n s were m e a s u r e d b y t h e m e t h o d o f B u r t o n [ 2 ] . The S-100 p r o t e i n was p r e p a r e d a c c o r d i n g t o the p r o c e d u r e o f M o o r e [ 1 4 ] . a - A m a n i t i n was a g e n e r o u s gift o f Dr. DiMauro, University o f R o m e . Initially the stimulating a c t i o n o f t h e p r o t e i n was tested o n s o n i c a t e d nuclei (Fig. 2). In a c c o r d a n c e with t h e d a t a o f D u t t o n and Mahler [ 6 ] , t h e R N A - p o l y m e r a s e activity appears to be l o w e r in d i s r u p t e d nuclei t h a n in i n t a c t ones, b u t t h e S-100 also stimulates t h e e n z y m e activity u n d e r these c o n d i t i o n s . T h e R N A - p o l y m e r a s e activity was t h e n tested d i r e c t l y in isolated nucleoli in t h e presence or in t h e absence o f t h e S-100. Fig. 3 shows t h a t o u r nucleolar p r e p a r a t i o n s exhibit R N A s y n t h e t i c activity w h i c h is also s t i m u l a t e d b y t h e S-100 protein. As previously observed with isolated brain nuclei [ 1 1 ] , the stimulating effect o f S-100 was c o n f i r m e d in t h e presence o f a - a m a n i t i n .
25
30
Z
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÷
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Sonicated nuclei
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15
time (mini
Fig. 2. Effect of the S-100 protein on the Mg~+-dependent RNA-polymerase activity in sonicated brain nuclei isolated from newborn rat. Sonicated nuclei (four times for 10 sec each time at 10 sec intervals at 1.5 A) were preincubated for 10 rain at 37°C in 100 mM tris-HC1 buffer pH 8.0 containing 4 mM MgC12, in the presence or in the absence of S-100 protein (200 ~g/ml). The assay was started in the reaction mixture at 37°C with 0.1 ml o f preincubated suspension, and was stopped after 15 rain with 5 ml of ice-cold perchloric acid (0.5 N) in 1% sodium pyrophosphate. One milligram of bovine serum albumin was added as a carrier. The precipitate was then washed twice more with 6 ml of cold perchloric acid (0.2 N) in 1% sodium pyrophosphate. The final precipitate was solubilized in 0.5 ml of NCS reagent (Nuclear Chicago Solubilizer) and 10 ml of toluene-based scintillation liquid were added for counting the radioactivity in a Nuclear Chicago scintillation spectrometer. Counting efficiency was approximately 35%. The activity was expressed in distint./min/mg DNA. D N A per assay tube: approximately 200 ug. The values are the average o f three experiments. Fig. 3. Time course of Mg2+-activated RNA-polymerase reaction in isolated brain nucleoli from newborn rat. The e n z y m e activity was determined as described in Fig. 2 for the indicated times. D N A per assay tube: 50 ~g. The values are the average o f four experiments.
This finding is obvious in isolated nucleoli, since ~-amanitin is a selective inhibitor of the nucleoplasmic RNA-polymerase activity. On the contrary, no stimulating activity was observed in the presence of bovine serum albumin, poly-/-aspartate (M.W. 4870) or poly-l-glutamate (M.W. 19,700) employed as a control {not shown). The above data give additional information about the properties of the
I
26 nuclear f r a c t i o n o f S-100, b u t at present it is impossible t o state to w h a t ext e n t these in vitro findings m a y be c o r r e l a t e d to t h e in vivo behavior o f the protein. With these limitations in mind, the f o l l o w i n g c o n s i d e r a t i o n s m a y be m a d e : (1) the integrity of t h e nuclear s t r u c t u r e is n o t a prerequisite for the a c t i o n o f the p r o t e i n ; (2) the S-100 p r o t e i n does n o t e x e r t its s t i m u l a t o r y activity m e r e l y b y e n h a n c i n g the e n t r a n c e into the nucleus o f R N A precursors; (3) the S-100 p r o t e i n is able to stimulate directly n a k e d nucleoli, alt h o u g h t h e possibility for c o o p e r a t i v e actions b y n u c l e o p l a s m i c a n d / o r nuclear m e m b r a n o u s c o m p o n e n t s in i n t a c t nuclei c a n n o t be ruled out. Since nucleoli are believed t o lack tissue specificity [ 1 9 ] , it is intriguing t h a t t h e y are s t i m u l a t e d b y a brain-specific protein, w h i c h f u r t h e r m o r e affects specifically brain nuclei. ACKNOWLEDGEMENTS T h e a u t h o r s are m u c h i n d e b t e d to Prof. N. Miani for critical review o f the m a n u s c r i p t , and t o Dr. C. Olivieri-Sangiacomo f o r M/E e x a m i n a t i o n s o f nucleolar preparations. This investigation was partially s u p p o r t e d b y C.N.R. C o n t r a c t No. 74.00229.04. REFERENCES 1 Benda, P., Lightbody, J., Sato, G., Levine, L., and Sweet, W.A., Differentiated rat glial cell strain in tissue culture, Science, 161 (1968) 370--371. 2 Burton, K., A study or" the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid, Biochem. J., 62 (1956) 315--323. 3 Busch, H., Isolation and purification of nucleoli. In L. Grossman and K. Moldave (Eds.), Methods in Enzymology, Volume XII, Part A, Academic Press, New York, 1967, pp. 448--464. 4 Cicero, T.J., Cowan, W.M., Moore, B.W., and Suntzeff, V., The cellular localization of the two brain specific proteins S-100 and 14.3.2, Brain Res., 18 (1970) 25--34. 5 Donato, R., Michetti, F., and Miani, N., Soluble and membrane-bound S-100 protein in cerebral cortex synaptosomes. Properties of the S-100 receptor, Brain Res., 98 (1975) 561--573. 6 Dutton, G.H., and Mahler, H.R., In vitro RNA synthesis by intact rat brain nuclei, J. Neurochem., 15 (1968) 765--780. 7 Haglid, K., and Stavrou, D., Water-soluble and pentanol-extractable proteins in human brain tissue and human brain tumours, with special reference to S-100 protein, J. Neurochem., 20 (1973) 1523--1532. 8 Hyd~n, H., and McEwen, B.S., A glial protein specific for the nervous system, Proc. nat. Acad. Sci. (Wash.), 55 (1966) 354--358. 9 L~bvtrup-Rein, H., and McEwen, B.S., Isolation and fractionation of rat brain nuclei, J. Cell Biol., 30 (1966) 405--415. 10 Miani, N., DeRenzis, G., Michetti, F., Correr, S., Olivieri-Sangiacomo, C., and Caniglia, A., Axonal transport of S-100 protein in mammalian nerve fibres, J. Neurochem., 19 (1972) 1387--1394. 11 Miani, N., Michetti, F., DeRenzis, G., and Caniglia, A., Effect of a brain specific protein
27
12 13
14 15 16 17 18 19
(S-100 protein) on the nucleolar RNA-polymerase activity in isolated brain nuclei, Experientia (Basel), 29 (1973) 1499--1501. Michetti, F., Miani, N., DeRenzis, G., Caniglia, A., and Correr, S., Nuclear localization of S-100 protein, J. Neurochem., 22 (1974) 239--244. Montanaro, N., Novello, F., and Stirpe, F., Effect of ~-amanitin on ribonucleic acid polymerase II of rat brain nuclei and on retention of avoidance conditioning, Biochem. J., 125 (1971) 1087--1090. Moore, B.W., A soluble protein characteristic of the nervous system, Biochem. biophys. Res. Commun., 19 (1965) 739--744. Packman, P.M., Blomstrand, C., and Hamberger, A., Disc electrophoretic separation of proteins in neuronal, glial, and subcellular fractions from cerebral cortex, J. Neurochem., 18 (1971) 479--487. Rusca, G., Calissano, P., and Alem~, S., Identification of a membrane-bound fraction of the S-100 protein, Brain. Res., 49 (1972) 223--227. Schubert, D., Heinemann, S., Carlish, W., Tarikas, H., Kimes, B., Patrick, J., Steinbach, J.H., Culy, W., and Brandt, B.L., Clonal cell lines from rat central nervous system, Nature (Lond.), 249 (1974) 224--227. Sviridov, S.M., Korochin, L.I., Ivanov, V.N., Maletskaya, E.I., and Bakhtina, T.K., Immunohistochemical studies of S-100 protein during post-natal ontogenesis of the brain of two rat strains, J. Neurochem., 19 (1972) 713--718. Takahashi, Y., Araki, K., Ikeda, K., and Oyanagi, S., Isolation and some characteristics of nucleoli from the brain, Brain Res., 73 (1974) 189--203.