Brain Research, 109 (1976) 649-655 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
649
Soluble and membrane-bound S-100 protein in rat cerebral cortex $ynaptosomes during postnatal development
ROSARIO DONATO Department of Anatomyi~Universit~ Cattolica S. Cuore, 00168 Rome (Italy)
(Accepted March 2nd, 1976)
The brain-specific S-100 protein a2 is a glial 5,9,27,a5 as well as a neuronal la-15, 21,a0,a4,a~ protein which haslbeen implicated in neurophysiological functions s,1~,21, al,aa and behavioral parameters~5, ~8. As a soluble protein, S-100 accumulates at an extremely rapid rate in the mouse nervous system during the second and third postnatal week 1°, and in rat cerebrum after the second week 24. Since S-100 is also present in a membrane-bound form la-15,2~-~a,a6, changes in the membrane-bound fraction of the protein during postnatal development have been looked for. A brief account on this topic has appeared la. Homogenates and synaptosomes were obtained from the cerebral cortex of Wistar rats ranging in age from 1 to 60 days, as described by Autilio et al. a, and Gonatas et al.lL All operations were performed at 2-4 °C. The entire forebrain anterior to the colliculi was used in the case of the 1- and 8-day-old rats. The crude mitochondrial fraction was washed twice with 0.32 M sucrose in order to minimize the microsomal contamination 20. The synaptosomal fraction was, for all but 1-dayold rats, that recovered between 7.5 7o and 13 % Ficoll; in the case of newborn rats, the two fractions between 0.32 M sucrose and 5 7o Ficoll, and between 5 % and 7.5 70 Ficoll respectively, were used 19. Each synaptosomal fraction was diluted with 4 volumes of 0.32 M sucrose and centrifuged at 40,000 × g for 60 min. The pellet was washed once under the same conditions. Aliquots of the homogenate and synaptosomes were resuspended in Tris.HC1 5 mM, p H 8.5 (6 ml/g fresh tissue) and kept at 4 °C for 90 min for hypo-osmotic shock 11. The suspensions were then stored at - - 2 0 °C until use. Aliquots of the stored material were thawed and centrifuged at 105,000 × g for 60 min. The pellet was resuspended in cold distilled water (6 ml/g fresh tissue) and centrifuged as before. The treatment was repeated 3 more times, i.e., until no more S-100 could be detected immunologically in the supernatant. The supernatants were referred to as the water-released fractions. The water-released fraction was considered under all aspects together with the cytosol (105,000 × g supernatant of the homogenate) in the case of homogenates, while it was referred to as the soluble fraction of synaptosomes in the case of synaptosomal fractions. The resultant pellet was resuspended in 59/00n-pentanol, Tris.HC1 10 mM, pH 7.4 (6 ml/g fresh tissue) and kept
650 under vigorous shaking overnight in the cold room. The suspension was centrifuged as before and the pellet was washed once under the same conditions. The two supernatants were referred to as the n-pentanol extract. Proteins extracted with n-pentanol were considered to be membrane-bound, n-Pentanol was chosen because it was shown to be very effective in extracting S-10015,36. The soluble and solubilized fractions were subjected to complement fixation assay by the method of Moore and Perez a3 as described elsewhere 14,1~. S-100 was obtained from ox brain as described by Moore 3~. Protein was measured as described by Lowry et aL es against a standard solution of bovine serum albumin. Acetylcholinesterase (ACHE) and choline acetyltransferase (ChAc) activities were measured according to the methods of Fonnum 17,18 ([acetyl-l-14C]choline chloride (Amersham), spec. act. 11.8 mCi/mmole; [acetyl1-14C]coenzyme A (Amersham), spec. act. 58 mCi/mmole). The cerebral cortex from very young rats (up to 10 days after birth) contains mainly neuronal elements and, to a much lesser extent, glial elements 1,2,6,7,16,29. Synaptogenesis seems to reach adult values around the 20-25th postnatal day, with a sharp increase in the number of synaptic junctions between days 12 and 20 (ref. 1). Accordingly, the synaptosomal fraction frc,n newborn rat cerebral cortex contains a limited number of synaptosomes, the emaining membranous profiles belonging to other neuronal structures 19. The pe centage of well-defined synaptosomes in the classical synaptosomal band of the gradient increases as a function of age 19; thus, any finding of the present report should be evaluated in the light of these limits. However, two enzymic activities: that of AChE (generally neuronal) and that of ChAc (primarily synaptosomal) can be used as an index of the synaptosomal population of the gradient fractions. The results indicate that the rate of increase of the enzymic activities are similar; both show a higher rate of increase between days 15 and 21, paralleling the increase of synaptosomal structures. Tables I and II show the distribution of S-100 protein in the homogenate and synaptosomes of adult rat cerebral cortex. S-100 is mainly a soluble protein in both fractions, but a consistent amount of it is associated with membranes. It is confirmed that the bound/soluble S-100 ratio is much greater for synaptosomes than for whole homogenate ~5 (Table II). About 2 % of the total S-100 content of rat cerebral cortex is associated with synaptosomes (Table I). This value greatly underestimates the TABLE I S - 1 0 0 CONTENT (,teg
S-100/g FRESH
TISSUE) IN HOMOGENATE AND IN SYNAPTOSOMES FROM 60-DAY-OLD
RAT CEREBRAL CORTEX
Membrane-bound S-100 was the n-pentanol extracted fraction of the protein. Values are the mean of three triplicate experiments. Maximal S.E.M. was not more than 10%.
Homogenate Synaptosomes
Soluble
Membranebound
%
171.76 3.12
35.02 1.48
100 2.21
651 TABLE II P E R C E N T A G E D I S T R I B U T I O N OF S O L U B L E A N D CEREBRAL CORTEX HOMOGENATE AND
MEMBRANE-BOUND
S-100
PROTEIN IN 6 0 - d A Y - O L D
RAT
SYNAPTOSOMES
Figures were calculated from those reported in Table I.
Soluble Membrane-bound Bound/soluble x 100
Homogenate
Synaptosomes
83.07 16.93 20.39
67.93 32.07 47.20
actual S-100 content of nerve endings, since the yield of synaptosomes isolated on a Ficoll-sucrose gradient is rather poor 4. It can be reasonably excluded that synaptosomal S-100 results from contamination from cytosol S-100 during the isolation procedure14,15. On the contrary, some of the soluble fraction of S- 100 of synaptosomes could be lost during the isolation of nerve endings. S-100 is detectable in rat cerebral cortex homogenate both in the soluble and membrane-bound forms as early as the first postnatal day, and accumulates at a rapid rate during the third and fourth week after birth (Fig. 1). S-100 concentration (#g S-100/mg soluble or membrane protein) also increases as a function of age (Fig. 2). This is good evidence of a net increase of the S-100 content in developing cerebral cortex. Membrane-bound S-100 represents about 17~ of the total S-100 content by the end of the first postnatal week (Fig. 3). Before this age, S-100 is mainly a membrane-bound protein (Fig. 3). Whether this is due to the different S-100 species a7 or to variations of the membrane milieu during development or to the different cellular
~s0
=
1
i
6
i
15
i
21
~ Days
Fig. 1. Pattern of accumulation of soluble and membrane-bound S-100 protein, and AChE and
ChAc activities in rat cerebral cortex homogenate during postnatal development. Values, which are the mean of two or three triplicate experiments, are expressed as percentage of maximum. O, total S-100 content/g fresh tissue (maximum at 60 days = 206.68 /~g); D, soluble S-100/g fresh tissue (maximum at 60 days = 171.76/~g); A, membrane-bound S-100/g fresh tissue (maximum at 60 days = 35.02/~g), O, AChE activity (maximum at 60 days = 10.14/~moles/rng protein/60 min); m , ChAc activity (maximum at 60 days = 0.0234/~moles/mg protein/60 min).
652
"5
50
•
1C B~ ' ° 1
8
,
~
,
15
21
30 Days
60
Fig. 2. S-I00 concentration (pg S-100/mg protein) in homogenate and in synaptosomes of rat cerebral cortex during postnatal development. Values, which are the mean of two or three triplicate experiments, are expressed as percentages of maximum. D, S-100 concentration in the soluble fraction of the homogenate (maximum at 60 days = 4.44); ~ , S-100 concentration in the membranous fraction of the homogenate (maximum at 60 days = 0.90); ~ S,100 concentration in the soluble fraction of synaptosomes (maximum at 60 days = 2.79); A , S-100 concentration in the membranous fraction of synaptosomes (maximum at 21 days = 0.88).
composition of cerebral cortex during the first postnatal week with respect to older ages 1,2,6,7,16,29 remains to be clarified. In any event, membranes seem to acquire immediately the ability to incorporate S-100. Fig. 1 also shows that both AChE and ChAc activities increase as a function of age. Many of the considerations made on soluble and membrane-bound S-100 for the cerebral cortex homogenate apply to cerebral cortex synaptosomes (Figs. 2 and 4). However, a few points should be separately considered: (1) the bound/ soluble S-100 ratio is significantly greater for synaptosomes than for whole homogenate by the end of the first postnatal week (Fig. 3); (2) the maximal increment of
E .~ 5O
1
8
15
21
30 Days
60
Fig. 3. Bound/soluble S-100 ratio in homogcnate ( ~ ) (maximum at birth -- 142.58) and in synaptosomes ( A ) (maximum at birth = 118,00)from rat cerebral cortex during postnatal d e v e l 0 ~ n t . Bound S-100 was the n-pentanol extracted fraction of the protein.
653
E
"15
~c
Fig. 4. Pattern of accumulation of soluble and membrane-bound S-100 protein, and AChE and ChAc activities in rat cerebral cortex synaptosomes during postnatal development. Values, which are the mean of two or three triplicate experiments, are expressed as percentages of maximum. (3, total S-100 content/g fresh tissue (maximum at 60 days = 4.60/~g); IS], soluble S-100/g fresh tissue (maximum at 60 days = 3.12/~g); A, membrane-bound S-100/g fresh tissue (maximum at 60 days = 1.48/~g); e , AChE activity (maximum at 60 days = 10.59/~moles/mg protein/60 min); , , ChAc activity (maximum at 60 days = 0.245/Lmoles/mg protein/60 min). both soluble and membrane-bound fractions of S-100 in synaptosomes is registered during the second and third postnatal week (Fig. 4), i.e., in coincidence with the maximal increment in the number of synapses z, and of AChE and ChAc activities; (3) the highest membrane-bound S-100 concentration in synaptosomes is observed at the end of the third postnatal week (Fig. 2), i.e., when the number of synaptic junctions has reached adult values 1. Point (1) suggests that, as glial elements proliferate and neuronal structures differentiate, a more definite pattern of distribution of S-100 could take place. It could be speculated that synaptosomal particulates are a rather privileged site of interaction lor S-100 (see ref. 15) and/or membrane-bound S-100 is predominantly neuronal in distribution. Points (2) and (3) indicate that the accumulation of both soluble and membrane-bound S-100 fractions parallels synaptogenesis, and that a great amount of other proteins, in addition to S-100, is incorporated into synaptosomal particulates after the period of maximal increment in the number of synapses. The author is much indebted to Prof. N. Miani for useful suggestions during the course of experimentation and for critical review of the manuscript. Dr. G. De Renzis kindly measured the AChE and ChAc activitities. This work was partially supported by C.N.R. Contracts 71.00856.04 and 72.00817.04.
1 AGHAJANIAN,G. D., ANDBLOOM,F. E., The formation of synaptic junction in developing rat brain. A quantitative electron microscopic study, Brain Research, 6 (1967) 716-727. 2 ALTMAN,J., AND DA$, G. D., Postnatal origin of microncurons in the rat brain, Nature (Lond.), 207 (1965) 953-956,
654 3 AUTILIO, L. A., APPEL, S. H., PETTIS, P., AND GAMBETTI, P.-U, Biochemical studies of synapses in vitro. I. Protein synthesis, Biochemistry, 7 (1968) 2615-2622. 4 BARONDES,S. H., Synaptic macromolecules: identification and metabolism, Ann. Rev. Biochem., 43 (1974) 147-168. 5 BENDA, P., LIGHTBODY,J., SATO, G., LEVINE,L., AND SWEET, W., Differentiated glial cell strain in tissue culture, Science, 161 (1968) 370-371. 6 BURDMAN,J. A., The relationship between D N A synthesis and the synthesis of nuclear proteins in rat brain during development, J. Neurochem., 19 (1972) 1459-1469. 7 CALEY, D. W., AND MAXWELL, D. S., An electron microscopic study of neurons during postnatal development of the rat cerebral cortex, J. comp. Neurol., 133 0968) 17-43. 8 CALISSANO, P., AND BANGHAM, A. D., Effect of two brain specific proteins (S-100 and 14.3.2) on cation diffusion across artificial membranes, Biochem. biophys. Res. Commun., 43 (1971) 504-509. 9 CICERO,T. J., COWAN, W. M., MOORE, B. W., AND SUNTZEFE, V., The cellular localization of the two brain specific proteins S-t00 and 14.3.2, Brain Research, 18 (1969) 25-34. 10 CICERO, T. J., FERRENDELLI,J. A., SUNTZEFF,V., AND MOORE, B. W., Regional changes in CNS levels of the S-100 and 14.3.2 proteins during development and aging of the mouse, J. Neurochem., 19 (1972) 2119-2125. 11 COTMAN, C. W., AND MATTHEWS,D. A., Synaptic plasma membranes : isolation and partial characterization, Biochim. biophys. Acta (Amst.), 249 (1971) 380-394. 12 DE ROBERTIS, E., Ultrastructure and cytcchemistry of the synaptic region, Science, 156 (1967) 907-914. 13 DONATO, R., Membrane bound S-100 protein in cerebral cortex synaptosomes during development, Exp. Brain Res., Suppl., 23 (1975) 61. 14 DONATO,R., AND MICHETTI, F., S-100 protein in cerebral cortex synaptosomes, Experientia (Basel), 30 (1974) 511-512. 15 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 Research, 98 (1975) 561-573. 16 EAYRS,J. T., AND GOODHEAD,B., Postnatal development of the cerebral cortex in the rat, J. Anat. (Lond.), 93 (1959) 385-402. 17 FONNUM, F., Radiochemical microassays for the determination of choline acetyltransferase and acetylcholinesterase activities, Biochem. J., 115 (1969) 465-472. 18 FONNUM,F., Surface charge of choline acetyltransferase from different species, J. Neurochem., 17 (1970) 1095-1100. 19 GONATAS,N. K., AUTILIO-GAMBETTI,L., GAMBETTI,P. L., AND SHAFER, B., Morphological and biochemical changes in rat synaptosome fractions during neonatal development, J. Cell BioL, 51 (1971) 484498. 20 GURD, J. W., JONES,L. R., MAHLER,H. R., AND MOORE,W. J., Isolation and partial characterization of rat brain synaptic plasma membranes, J. Neurochem., 22 (1974) 281-290. 21 HAGLID, K. G., HAMBERGER,A., HANSSON,H. A., HYDEN, H., PERSSON, L., AND R()NNBACH, L., S- 100 protein in synapses of the central nervous system, Nature (Lond.), 25 (1974) 532-534. 22 HAGLID,K. G., AND STAVROU,D., Water soluble and pentanol extractable proteins in human brain normal tissue and human tumors, with special reference to S-100 protein, J. Neurochem., 20 (1973) 1523-1532. 23 HAGLID, K. G., STAVROU,D., RONNBACH,L., CARLSSON,C. A., AND WE1DENBACH,W., The S-100 protein in water-soluble and pentanol-extractable form in normal human brain and tumors of the human nervous system, J. neurol. Sci., 20 (1973) 103-111. 24 HERSCHMAN,H. R., LEVINE, L , AND DE VELLIS,J., Appearance of a brain specific antigen (S-100 protein) in the developing rat brain, J. Neurochem., 18 (1971) 629-633. 25 HYD~N, H., AND LANGE, P. W., Brain-cell protein synthesis specifically related to learning, Proc. nat. Acad. Sci. (Wash.), 65 (1970) 898-904. 26 HYDt~N,H., AND LANGE, P. W., Protein synthesis in limbic structures during change in behaviour, Brain Research,, 22 (1970) 423-425. 27 LIGHTBODY,J., PFEIFFER,S. E., KORNBLITH, P. L., AND HERSCHMAN,H. R., Biochemically differentiated clonal human glial cells in tissue culture, J. Neurobiol., 4 (1970) 41 t-417. 28 LOWRY,O. H., ROSEBROUGH,J. J., FARR, A. L., AND RANDALL, R. J., Protein measurement with the Folin phenol reagent, J. bioL Chem., 193 (1951) 265-275. 29 MCILWAIN, H., AND BACHELARD,H. S., Biochemistry and the Central Nervous System, 4th ed., Churchill Livingstone, Edinburgh, I971, pp. 406-444. 30 MIANI, N., DE RENZIS,G., MICHETTI,F., CORRER,S., OL1VIERI-SANGIACOMO,C., ANDCANIGLIA,A.,
655 Axonal transport of S-100 protein in mammalian nerve fibres, J. Neurochem., 19 (1972) 1387-1394. 31 MIANt,N., MXCHETTI,F., DE RENZlS, G., AND CANaGLtA,A., Effect of a brain specific protein (S-100 protein) on the nucleolar R N A polymerase activity in isolated brain nuclei, Experientia (Basel), 29 (1973) 1499-1501. 32 MOORE, B. W., A soluble protein characteristic of the nervous system, Biochem. biophys. Res. Commun., 19 (1965) 739-744. 33 MOORE, B. W., AND PEREZ, V. J., Complement fxation for antigens on a picogram level, J. Immunol., 96 (1966) 1000-1005. 34 PACKMAN,P. M., BLoMSrRAND, C., ANI) HAMBERGErt,A., Disc electrophoretic separation of proteins in neuronal, gliai and subcellular fractions from cerebral cortex, J. Neurochem., 18 (1971) 479-487. 35 PERZZ,V. J., OLr,mV, J. W., CICERO,T. J., MOORE,B. W., AND BA.N, B. A., Wallerian degeneration in rabbit optic nerve: cellular localization in the central nervous system of the S-100 and 14.3.2 proteins, J. Neurochem., 17 (1970) 511-519. 36 RUSCA, G., CALISSANO,P., AND ALEMA, S., Identification of a membrane-bound fraction of the S-100 protein, Brain Research, 49 (1972) 223-227. 37 SCHNEIDER,D. J., Nervous system proteins of wide phylogenetic distribution. In D. J. SCr~NEII)ER, R. H. AN6ELET'rl, R. A. BRAOSHAW,A. GRASSOAND B. W. MOORE(Eds.), Proteins of the Nervous System, Raven Press, New York, 1973, pp. 80-85. 38 SINGLE,V. B., AriD TALWAR,P. G., Identification of a protein fraction in the occipital cortex of the monkey rapidly labelled during the exposure of the animal to rhythmically flickering light, J. Neurochem., 16 (1969) 951-959. 39 ZUCKERMAN,J. E., HERSCHMAN,H. R., AND LEV1NE, L., Appearance of a brain specific antigen (the S-100 protein) during human foetal development, J. Neurochem., 17 (1970) 247-251.
649
Soluble and membrane-bound S-100 protein in rat cerebral cortex $ynaptosomes during postnatal development
ROSARIO DONATO Department of Anatomyi~Universit~ Cattolica S. Cuore, 00168 Rome (Italy)
(Accepted March 2nd, 1976)
The brain-specific S-100 protein a2 is a glial 5,9,27,a5 as well as a neuronal la-15, 21,a0,a4,a~ protein which haslbeen implicated in neurophysiological functions s,1~,21, al,aa and behavioral parameters~5, ~8. As a soluble protein, S-100 accumulates at an extremely rapid rate in the mouse nervous system during the second and third postnatal week 1°, and in rat cerebrum after the second week 24. Since S-100 is also present in a membrane-bound form la-15,2~-~a,a6, changes in the membrane-bound fraction of the protein during postnatal development have been looked for. A brief account on this topic has appeared la. Homogenates and synaptosomes were obtained from the cerebral cortex of Wistar rats ranging in age from 1 to 60 days, as described by Autilio et al. a, and Gonatas et al.lL All operations were performed at 2-4 °C. The entire forebrain anterior to the colliculi was used in the case of the 1- and 8-day-old rats. The crude mitochondrial fraction was washed twice with 0.32 M sucrose in order to minimize the microsomal contamination 20. The synaptosomal fraction was, for all but 1-dayold rats, that recovered between 7.5 7o and 13 % Ficoll; in the case of newborn rats, the two fractions between 0.32 M sucrose and 5 7o Ficoll, and between 5 % and 7.5 70 Ficoll respectively, were used 19. Each synaptosomal fraction was diluted with 4 volumes of 0.32 M sucrose and centrifuged at 40,000 × g for 60 min. The pellet was washed once under the same conditions. Aliquots of the homogenate and synaptosomes were resuspended in Tris.HC1 5 mM, p H 8.5 (6 ml/g fresh tissue) and kept at 4 °C for 90 min for hypo-osmotic shock 11. The suspensions were then stored at - - 2 0 °C until use. Aliquots of the stored material were thawed and centrifuged at 105,000 × g for 60 min. The pellet was resuspended in cold distilled water (6 ml/g fresh tissue) and centrifuged as before. The treatment was repeated 3 more times, i.e., until no more S-100 could be detected immunologically in the supernatant. The supernatants were referred to as the water-released fractions. The water-released fraction was considered under all aspects together with the cytosol (105,000 × g supernatant of the homogenate) in the case of homogenates, while it was referred to as the soluble fraction of synaptosomes in the case of synaptosomal fractions. The resultant pellet was resuspended in 59/00n-pentanol, Tris.HC1 10 mM, pH 7.4 (6 ml/g fresh tissue) and kept
650 under vigorous shaking overnight in the cold room. The suspension was centrifuged as before and the pellet was washed once under the same conditions. The two supernatants were referred to as the n-pentanol extract. Proteins extracted with n-pentanol were considered to be membrane-bound, n-Pentanol was chosen because it was shown to be very effective in extracting S-10015,36. The soluble and solubilized fractions were subjected to complement fixation assay by the method of Moore and Perez a3 as described elsewhere 14,1~. S-100 was obtained from ox brain as described by Moore 3~. Protein was measured as described by Lowry et aL es against a standard solution of bovine serum albumin. Acetylcholinesterase (ACHE) and choline acetyltransferase (ChAc) activities were measured according to the methods of Fonnum 17,18 ([acetyl-l-14C]choline chloride (Amersham), spec. act. 11.8 mCi/mmole; [acetyl1-14C]coenzyme A (Amersham), spec. act. 58 mCi/mmole). The cerebral cortex from very young rats (up to 10 days after birth) contains mainly neuronal elements and, to a much lesser extent, glial elements 1,2,6,7,16,29. Synaptogenesis seems to reach adult values around the 20-25th postnatal day, with a sharp increase in the number of synaptic junctions between days 12 and 20 (ref. 1). Accordingly, the synaptosomal fraction frc,n newborn rat cerebral cortex contains a limited number of synaptosomes, the emaining membranous profiles belonging to other neuronal structures 19. The pe centage of well-defined synaptosomes in the classical synaptosomal band of the gradient increases as a function of age 19; thus, any finding of the present report should be evaluated in the light of these limits. However, two enzymic activities: that of AChE (generally neuronal) and that of ChAc (primarily synaptosomal) can be used as an index of the synaptosomal population of the gradient fractions. The results indicate that the rate of increase of the enzymic activities are similar; both show a higher rate of increase between days 15 and 21, paralleling the increase of synaptosomal structures. Tables I and II show the distribution of S-100 protein in the homogenate and synaptosomes of adult rat cerebral cortex. S-100 is mainly a soluble protein in both fractions, but a consistent amount of it is associated with membranes. It is confirmed that the bound/soluble S-100 ratio is much greater for synaptosomes than for whole homogenate ~5 (Table II). About 2 % of the total S-100 content of rat cerebral cortex is associated with synaptosomes (Table I). This value greatly underestimates the TABLE I S - 1 0 0 CONTENT (,teg
S-100/g FRESH
TISSUE) IN HOMOGENATE AND IN SYNAPTOSOMES FROM 60-DAY-OLD
RAT CEREBRAL CORTEX
Membrane-bound S-100 was the n-pentanol extracted fraction of the protein. Values are the mean of three triplicate experiments. Maximal S.E.M. was not more than 10%.
Homogenate Synaptosomes
Soluble
Membranebound
%
171.76 3.12
35.02 1.48
100 2.21
651 TABLE II P E R C E N T A G E D I S T R I B U T I O N OF S O L U B L E A N D CEREBRAL CORTEX HOMOGENATE AND
MEMBRANE-BOUND
S-100
PROTEIN IN 6 0 - d A Y - O L D
RAT
SYNAPTOSOMES
Figures were calculated from those reported in Table I.
Soluble Membrane-bound Bound/soluble x 100
Homogenate
Synaptosomes
83.07 16.93 20.39
67.93 32.07 47.20
actual S-100 content of nerve endings, since the yield of synaptosomes isolated on a Ficoll-sucrose gradient is rather poor 4. It can be reasonably excluded that synaptosomal S-100 results from contamination from cytosol S-100 during the isolation procedure14,15. On the contrary, some of the soluble fraction of S- 100 of synaptosomes could be lost during the isolation of nerve endings. S-100 is detectable in rat cerebral cortex homogenate both in the soluble and membrane-bound forms as early as the first postnatal day, and accumulates at a rapid rate during the third and fourth week after birth (Fig. 1). S-100 concentration (#g S-100/mg soluble or membrane protein) also increases as a function of age (Fig. 2). This is good evidence of a net increase of the S-100 content in developing cerebral cortex. Membrane-bound S-100 represents about 17~ of the total S-100 content by the end of the first postnatal week (Fig. 3). Before this age, S-100 is mainly a membrane-bound protein (Fig. 3). Whether this is due to the different S-100 species a7 or to variations of the membrane milieu during development or to the different cellular
~s0
=
1
i
6
i
15
i
21
~ Days
Fig. 1. Pattern of accumulation of soluble and membrane-bound S-100 protein, and AChE and
ChAc activities in rat cerebral cortex homogenate during postnatal development. Values, which are the mean of two or three triplicate experiments, are expressed as percentage of maximum. O, total S-100 content/g fresh tissue (maximum at 60 days = 206.68 /~g); D, soluble S-100/g fresh tissue (maximum at 60 days = 171.76/~g); A, membrane-bound S-100/g fresh tissue (maximum at 60 days = 35.02/~g), O, AChE activity (maximum at 60 days = 10.14/~moles/rng protein/60 min); m , ChAc activity (maximum at 60 days = 0.0234/~moles/mg protein/60 min).
652
"5
50
•
1C B~ ' ° 1
8
,
~
,
15
21
30 Days
60
Fig. 2. S-I00 concentration (pg S-100/mg protein) in homogenate and in synaptosomes of rat cerebral cortex during postnatal development. Values, which are the mean of two or three triplicate experiments, are expressed as percentages of maximum. D, S-100 concentration in the soluble fraction of the homogenate (maximum at 60 days = 4.44); ~ , S-100 concentration in the membranous fraction of the homogenate (maximum at 60 days = 0.90); ~ S,100 concentration in the soluble fraction of synaptosomes (maximum at 60 days = 2.79); A , S-100 concentration in the membranous fraction of synaptosomes (maximum at 21 days = 0.88).
composition of cerebral cortex during the first postnatal week with respect to older ages 1,2,6,7,16,29 remains to be clarified. In any event, membranes seem to acquire immediately the ability to incorporate S-100. Fig. 1 also shows that both AChE and ChAc activities increase as a function of age. Many of the considerations made on soluble and membrane-bound S-100 for the cerebral cortex homogenate apply to cerebral cortex synaptosomes (Figs. 2 and 4). However, a few points should be separately considered: (1) the bound/ soluble S-100 ratio is significantly greater for synaptosomes than for whole homogenate by the end of the first postnatal week (Fig. 3); (2) the maximal increment of
E .~ 5O
1
8
15
21
30 Days
60
Fig. 3. Bound/soluble S-100 ratio in homogcnate ( ~ ) (maximum at birth -- 142.58) and in synaptosomes ( A ) (maximum at birth = 118,00)from rat cerebral cortex during postnatal d e v e l 0 ~ n t . Bound S-100 was the n-pentanol extracted fraction of the protein.
653
E
"15
~c
Fig. 4. Pattern of accumulation of soluble and membrane-bound S-100 protein, and AChE and ChAc activities in rat cerebral cortex synaptosomes during postnatal development. Values, which are the mean of two or three triplicate experiments, are expressed as percentages of maximum. (3, total S-100 content/g fresh tissue (maximum at 60 days = 4.60/~g); IS], soluble S-100/g fresh tissue (maximum at 60 days = 3.12/~g); A, membrane-bound S-100/g fresh tissue (maximum at 60 days = 1.48/~g); e , AChE activity (maximum at 60 days = 10.59/~moles/mg protein/60 min); , , ChAc activity (maximum at 60 days = 0.245/Lmoles/mg protein/60 min). both soluble and membrane-bound fractions of S-100 in synaptosomes is registered during the second and third postnatal week (Fig. 4), i.e., in coincidence with the maximal increment in the number of synapses z, and of AChE and ChAc activities; (3) the highest membrane-bound S-100 concentration in synaptosomes is observed at the end of the third postnatal week (Fig. 2), i.e., when the number of synaptic junctions has reached adult values 1. Point (1) suggests that, as glial elements proliferate and neuronal structures differentiate, a more definite pattern of distribution of S-100 could take place. It could be speculated that synaptosomal particulates are a rather privileged site of interaction lor S-100 (see ref. 15) and/or membrane-bound S-100 is predominantly neuronal in distribution. Points (2) and (3) indicate that the accumulation of both soluble and membrane-bound S-100 fractions parallels synaptogenesis, and that a great amount of other proteins, in addition to S-100, is incorporated into synaptosomal particulates after the period of maximal increment in the number of synapses. The author is much indebted to Prof. N. Miani for useful suggestions during the course of experimentation and for critical review of the manuscript. Dr. G. De Renzis kindly measured the AChE and ChAc activitities. This work was partially supported by C.N.R. Contracts 71.00856.04 and 72.00817.04.
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