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Fast and slow components of S-100 protein fraction: regional distribution in bovine central nervous system
SHORT COMMUNICATIONS
475
Fast and slow components of S-100 protein fraction: regional distribution in bovine central nervous system The neuroglial localization of S-100, a protein specific to nervous tissuea2,13, has been clearly established 1 3,5,12,16. Earlier reports suggesting a concomitant neuronal localization 11,15 were invalidated by more recent investigations 5,6,16. The S-100 protein fraction (F-S 100), hitherto considered as a homogeneous protein, from several mammalian species consists of two immunologically related fractions called fast (FMC) and slow (SMC) migrating components, both specific to nervous tissue 19 and both present in different, but constant, proportions in all areas of the central nervous system (CNS) 7. We hoped that this study on the regional distribution of FMC and SMC would give some indication of a preferential cellular localization of each component and thus suggest an explanation for the molecular heterogeneity in terms of 'isoproteins' synthesized by different cell types. F-S100, F M C and SMC were measured by quantitative densitometry of electropherograms on mixed agarose acrylamide gel slabs 18. Dissection of beef CNS, tissue homogenization and extraction of soluble proteins were described elsewhere 7. Proteins were determined according to Lowry et al. ~ . Both FMC and SMC were present in markedly different amounts in all the 21 areas of beef CNS examined (Tables I and II). The amounts of F-S100 in white matter were higher than, or equal to, those in gray matter (Tables I and III), as would be expected if oligodendrocytes were the cells richest in S-100. This seems to be true since tumoral oligodendrocytes at the steady state contained 2.5 times more S-100 protein than tumoral astrocytes 1. It also seems to be so for normal nervous tissue where a strong immunofluorescence in oligodendrocyte cytoplasm was observed, as well as scattered fluorescence 'within the membranous system of glial cells'lL The observation agreed with histochemical findings suggesting that, in a given area, oligodendrocytes had more oxidative enzymic activity than normal astrocytes 9. The levels of metabolism of glial cells, however, also depended on the region in which they were located as indicated by an increasing rostro-caudal gradient of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) 4, an enzyme that in white matter was mainly in glial cells 10. A rostro-caudal gradient of increasing amounts of F-S100 from cerebral cortex to spinal cord was discernible (Tables I and II). This gradient was also evident from other data on adult 14,21 and fetal2, 21 human CNS where the regions with the highest levels in S-100 antigen were also those in which the antigen appeared first. The two areas richest in F-S100, the optical tract and cerebral pedunculi, were exceptions to the gradient rule. The optical tract has been reported to possess an unusually high activity of lactic dehydrogenase (EC 1.1.1.27) 17, a soluble cytoplasmic enzyme, which, since it appears to be present only in small amounts within axons, can be taken, in a nerve tract, as an indication of the size of the glial cell population 1°. F M C also followed a rostro-caudal concentration gradient except in the quadrigenal bodies, optical tract, and cerebral pedunculi. SMC did not follow a strict Brain Research, 26 (1971) 475-479
476
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Brain Research, 26 (1971) 475-479
478
SHORT COMMUNICATIONS
TABLE III RATIOS
BETWEEN
GRAY
AND
WHITE
MATTER
LEVELS
OF
F-S100, FMC
AND
SMC or
DIFFERENT
CNS
REGIONS
The ratios were calculated from the amounts expressed per mg total soluble proteins in columns A, and per g tissue (wet weight) in columns B. C N S region
Whole F-SIO0 A
FMC B
A
Bovine
Human* Bovine
Human** Bovine
0.88 0.75 0.64 0.60
---0.55
-0.96 -0.34 to 0.25***
SMC B
A
B
Bovine • 7¸
Cervical spinal cord Cerebellum Hippocampus Cerebral cortex
1.11 1.05 0.68 0.81
0.72 1.02 0.55 1.12
0.92 1.42 0.58 1.51
1.22 0.34 0.81 0.26
1.53 0.48 0.86 0.36
* According to BendaL ** Calculated from the results of Moore et alfl 4. *** Depending on the cortical area.
a n a t o m i c a l gradient a l t h o u g h the lowest levels were in the various gray matters (except spinal gray matter) a n d the highest in the optical tract a n d cerebral pedunculi. It is clear that the findings presented here, besides being in general agreement with a glial localization of b o t h c o m p o n e n t s of F-S100, do n o t allow any definite conclusion c o n c e r n i n g a m o r e specific cellular localization either o f F M C or of SMC. However we could tentatively relate F M C to oligodendrocytes a n d S M C to astrocytes o n the basis of the following rather t e n u o u s evidence. I n cerebral pedunculi, in which 30 ~ of F-S100 was S M C 7, 35 ~ of the glial cells c o n t a i n e d no D P N H 2 - d i a p h o r a s e (EC 1.6.99.1), considered as a m a r k e r for oligodendrocytes in n e r v o u s tracts s. The equivalent a m o u n t s of S M C a n d F M C in the optical tract m i g h t be explained by the reported presence of a particular type of astrocyte (rich in extensive processes) together with typical astrocytes a n d oligodendrocytes 2°.
Centre de Neurochimie du C N R S , Institut de Chimie Biologique, Facultd de M~deeine, 67 Strasbourg (France)
G. GOMBOS* W. FILIPOWICZ G. VINCENDON
1 BENDA,P., Prot6ine S-100 et cellules gliales du rat, Rev. neurol., 118 (1968) 364-367. 2 BENDA,P., Prot6ine S-100 et tumeurs c6r6brales humaines, Rev. neurol., 118 (1968) 368-372. 3 BENDA,P., LmHTBODY,J., SATO,G., LEVINE,L., ANDSWEET,W., Differentiated rat glial cell strain in tissue culture, Science, 161 (1969) 370-371. 4 BL~LL, M. V., LOWRY, O. H., ROBERTS,N. P ' CHANG, M. L. W., AND KAPPHAHN,J. I., The quantitative histochemistry of the brain. V. Enzymes of glucose metabolism, J. bioL Chem., 232 (1958) 979-993. * Attach6 de Recherche au C.N.R.S. Brain Research, 26 (1971) 475-479
479
SHORT COMMUNICATIONS
5 CICERO,T. J., COWAN,W. M., MOORE, B. W., AND SUNTZEFF,V., The cellular localization for the two brain specific proteins, S-100 and 14-3-2, Brain Research, 18 (1970) 25-34. 6 DRAVID, A. R., AND BURDMAN, J. A., Acidic proteins in rat brain nuclei: disc-electrophoresis, J. Neurochem., 15 (1968) 25-30. 7 FILiPOWlCZ, W., VINCENDON, G., MANDEL, P., AND GOMBOS, G., Topographical distribution of fast and slow migrating fraction of beef brain S-100 protein fraction, Life Sci., 7 (1968) 1243-1250. 8 FRIEDE,R. L., A histochemical study of DPN-Diaphorase in human white matter with some notes on myelinisation, J. Neurochem., 8 (1961) 17-30. 9 FRIEDE, R. L., Enzyme. Histochemistry of neuroglia. In E. D. P. DE ROBERTISAND R. CARREA (Eds.), Biology of Neuroglia, Progress in Brain Research, Vol. 15, Elsevier, Amsterdam, 1965, pp. 35-47. 10 FRIEDE, R. L., FLEMING, L. M., AND KNOLLER, M., A comparative mapping of enzymes involved in hexosemonophosphate shunt and citric acid cycle in the brain, ar. Neurochem., 10 (1963) 263277. 11 HYDI~N,H., AND McEwEN, B., A glial protein specific for the nervous system, Proc. nat. Acad. Sci. (Wash.), 55 (1966) 354-358. 12 LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., Protein measurement with the Folin Phenol reagent, J. biol. Chem., 193 (1951) 265-275. 13 MOORE, B. W., A soluble protein characteristic of the nervous system, Biochem. biophys, res. Commun., 19 (1965) 739-744. 14 MOORE, B. W., PEREZ, V. J., AND GEHRING, M., Assay and regional distribution of a soluble protein characteristic of the nervous system, J. Neurochem., 15 (1968) 265-272. 15 PEREZ, V. J., AND MOORE, B. W., Wallerian degeneration in rabbit tibial nerve: Changes in the amounts of the S-100 protein, J. Neurochem., 15 (1968) 971-977. 16 PERLE,V. J., OLNEY,J., CICERO,T. J., MOORE,B. W., AND BAHN, B. A., Wallerian degeneration in rabbit optic nerve: Research on cellular localization of the S-100 and 14-3-2 proteins, J. Neurochem., 17 (1970) 511-519. 17 STROMINGER,J. L., AND LOWRY, O. H., The quantitative histochemistry of the brain. IV. Lactic, malic and glutamic dehydrogenases, J. biol. Chem., 213 (1955) 635-646. 18 TARDY, J., UYEMURA, K., VINCENDON, G., ET GOMBOS, G., Une m6thode densitom6trique de d6termination quantitative de la fraction prot6ique S-100, Ann. Biol. clin., in press. 19 UYEMURA, K., TARDY, J., VINCENDON, G., MANDEL, P., ET GOMBOS, G., Mise en 6vidence de prot6ines sp6cifiques du cerveau chez les Mammif6res, C.R. Soc. Biol. (Paris), 161 (1967) 1396-1399. 20 WENDELL-SMITH,C. P., BLUNT, M. J., BALDWIN, F., AND BAISLEY,P. B., Neurone-satellite cell relationship, Nature (Lond.), 205 (1965) 781-782. 21 ZUCI~ERMAN,J, E., HERSCHMAN,J. R., AND LEVINE, L., Appearance of a brain specific antigen (the S-100 protein) during human foetal development, J. Neurochem., 17 (1970) 247-251. (Accepted Decemberl8th, 1970)
Brain Research, 26 (1971) 475-479
Erratum Brain Research, 26 (1971) 177-183 T h e m a g n i f i c a t i o n s o f F i g u r e s 1, 2 a n d 4 read ' × 'x
100';
t h e y should read
20, x 23 a n d x 18', r e s p e c t i v e l y .
Brain Research, 26 (1971) 479
475
Fast and slow components of S-100 protein fraction: regional distribution in bovine central nervous system The neuroglial localization of S-100, a protein specific to nervous tissuea2,13, has been clearly established 1 3,5,12,16. Earlier reports suggesting a concomitant neuronal localization 11,15 were invalidated by more recent investigations 5,6,16. The S-100 protein fraction (F-S 100), hitherto considered as a homogeneous protein, from several mammalian species consists of two immunologically related fractions called fast (FMC) and slow (SMC) migrating components, both specific to nervous tissue 19 and both present in different, but constant, proportions in all areas of the central nervous system (CNS) 7. We hoped that this study on the regional distribution of FMC and SMC would give some indication of a preferential cellular localization of each component and thus suggest an explanation for the molecular heterogeneity in terms of 'isoproteins' synthesized by different cell types. F-S100, F M C and SMC were measured by quantitative densitometry of electropherograms on mixed agarose acrylamide gel slabs 18. Dissection of beef CNS, tissue homogenization and extraction of soluble proteins were described elsewhere 7. Proteins were determined according to Lowry et al. ~ . Both FMC and SMC were present in markedly different amounts in all the 21 areas of beef CNS examined (Tables I and II). The amounts of F-S100 in white matter were higher than, or equal to, those in gray matter (Tables I and III), as would be expected if oligodendrocytes were the cells richest in S-100. This seems to be true since tumoral oligodendrocytes at the steady state contained 2.5 times more S-100 protein than tumoral astrocytes 1. It also seems to be so for normal nervous tissue where a strong immunofluorescence in oligodendrocyte cytoplasm was observed, as well as scattered fluorescence 'within the membranous system of glial cells'lL The observation agreed with histochemical findings suggesting that, in a given area, oligodendrocytes had more oxidative enzymic activity than normal astrocytes 9. The levels of metabolism of glial cells, however, also depended on the region in which they were located as indicated by an increasing rostro-caudal gradient of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) 4, an enzyme that in white matter was mainly in glial cells 10. A rostro-caudal gradient of increasing amounts of F-S100 from cerebral cortex to spinal cord was discernible (Tables I and II). This gradient was also evident from other data on adult 14,21 and fetal2, 21 human CNS where the regions with the highest levels in S-100 antigen were also those in which the antigen appeared first. The two areas richest in F-S100, the optical tract and cerebral pedunculi, were exceptions to the gradient rule. The optical tract has been reported to possess an unusually high activity of lactic dehydrogenase (EC 1.1.1.27) 17, a soluble cytoplasmic enzyme, which, since it appears to be present only in small amounts within axons, can be taken, in a nerve tract, as an indication of the size of the glial cell population 1°. F M C also followed a rostro-caudal concentration gradient except in the quadrigenal bodies, optical tract, and cerebral pedunculi. SMC did not follow a strict Brain Research, 26 (1971) 475-479
476
SHORT COMMUNICATIONS
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Brain Research, 26 (1971) 475-479
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477
SHORT COMMUNICATIONS
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Brain Research, 26 (1971) 475-479
478
SHORT COMMUNICATIONS
TABLE III RATIOS
BETWEEN
GRAY
AND
WHITE
MATTER
LEVELS
OF
F-S100, FMC
AND
SMC or
DIFFERENT
CNS
REGIONS
The ratios were calculated from the amounts expressed per mg total soluble proteins in columns A, and per g tissue (wet weight) in columns B. C N S region
Whole F-SIO0 A
FMC B
A
Bovine
Human* Bovine
Human** Bovine
0.88 0.75 0.64 0.60
---0.55
-0.96 -0.34 to 0.25***
SMC B
A
B
Bovine • 7¸
Cervical spinal cord Cerebellum Hippocampus Cerebral cortex
1.11 1.05 0.68 0.81
0.72 1.02 0.55 1.12
0.92 1.42 0.58 1.51
1.22 0.34 0.81 0.26
1.53 0.48 0.86 0.36
* According to BendaL ** Calculated from the results of Moore et alfl 4. *** Depending on the cortical area.
a n a t o m i c a l gradient a l t h o u g h the lowest levels were in the various gray matters (except spinal gray matter) a n d the highest in the optical tract a n d cerebral pedunculi. It is clear that the findings presented here, besides being in general agreement with a glial localization of b o t h c o m p o n e n t s of F-S100, do n o t allow any definite conclusion c o n c e r n i n g a m o r e specific cellular localization either o f F M C or of SMC. However we could tentatively relate F M C to oligodendrocytes a n d S M C to astrocytes o n the basis of the following rather t e n u o u s evidence. I n cerebral pedunculi, in which 30 ~ of F-S100 was S M C 7, 35 ~ of the glial cells c o n t a i n e d no D P N H 2 - d i a p h o r a s e (EC 1.6.99.1), considered as a m a r k e r for oligodendrocytes in n e r v o u s tracts s. The equivalent a m o u n t s of S M C a n d F M C in the optical tract m i g h t be explained by the reported presence of a particular type of astrocyte (rich in extensive processes) together with typical astrocytes a n d oligodendrocytes 2°.
Centre de Neurochimie du C N R S , Institut de Chimie Biologique, Facultd de M~deeine, 67 Strasbourg (France)
G. GOMBOS* W. FILIPOWICZ G. VINCENDON
1 BENDA,P., Prot6ine S-100 et cellules gliales du rat, Rev. neurol., 118 (1968) 364-367. 2 BENDA,P., Prot6ine S-100 et tumeurs c6r6brales humaines, Rev. neurol., 118 (1968) 368-372. 3 BENDA,P., LmHTBODY,J., SATO,G., LEVINE,L., ANDSWEET,W., Differentiated rat glial cell strain in tissue culture, Science, 161 (1969) 370-371. 4 BL~LL, M. V., LOWRY, O. H., ROBERTS,N. P ' CHANG, M. L. W., AND KAPPHAHN,J. I., The quantitative histochemistry of the brain. V. Enzymes of glucose metabolism, J. bioL Chem., 232 (1958) 979-993. * Attach6 de Recherche au C.N.R.S. Brain Research, 26 (1971) 475-479
479
SHORT COMMUNICATIONS
5 CICERO,T. J., COWAN,W. M., MOORE, B. W., AND SUNTZEFF,V., The cellular localization for the two brain specific proteins, S-100 and 14-3-2, Brain Research, 18 (1970) 25-34. 6 DRAVID, A. R., AND BURDMAN, J. A., Acidic proteins in rat brain nuclei: disc-electrophoresis, J. Neurochem., 15 (1968) 25-30. 7 FILiPOWlCZ, W., VINCENDON, G., MANDEL, P., AND GOMBOS, G., Topographical distribution of fast and slow migrating fraction of beef brain S-100 protein fraction, Life Sci., 7 (1968) 1243-1250. 8 FRIEDE,R. L., A histochemical study of DPN-Diaphorase in human white matter with some notes on myelinisation, J. Neurochem., 8 (1961) 17-30. 9 FRIEDE, R. L., Enzyme. Histochemistry of neuroglia. In E. D. P. DE ROBERTISAND R. CARREA (Eds.), Biology of Neuroglia, Progress in Brain Research, Vol. 15, Elsevier, Amsterdam, 1965, pp. 35-47. 10 FRIEDE, R. L., FLEMING, L. M., AND KNOLLER, M., A comparative mapping of enzymes involved in hexosemonophosphate shunt and citric acid cycle in the brain, ar. Neurochem., 10 (1963) 263277. 11 HYDI~N,H., AND McEwEN, B., A glial protein specific for the nervous system, Proc. nat. Acad. Sci. (Wash.), 55 (1966) 354-358. 12 LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., Protein measurement with the Folin Phenol reagent, J. biol. Chem., 193 (1951) 265-275. 13 MOORE, B. W., A soluble protein characteristic of the nervous system, Biochem. biophys, res. Commun., 19 (1965) 739-744. 14 MOORE, B. W., PEREZ, V. J., AND GEHRING, M., Assay and regional distribution of a soluble protein characteristic of the nervous system, J. Neurochem., 15 (1968) 265-272. 15 PEREZ, V. J., AND MOORE, B. W., Wallerian degeneration in rabbit tibial nerve: Changes in the amounts of the S-100 protein, J. Neurochem., 15 (1968) 971-977. 16 PERLE,V. J., OLNEY,J., CICERO,T. J., MOORE,B. W., AND BAHN, B. A., Wallerian degeneration in rabbit optic nerve: Research on cellular localization of the S-100 and 14-3-2 proteins, J. Neurochem., 17 (1970) 511-519. 17 STROMINGER,J. L., AND LOWRY, O. H., The quantitative histochemistry of the brain. IV. Lactic, malic and glutamic dehydrogenases, J. biol. Chem., 213 (1955) 635-646. 18 TARDY, J., UYEMURA, K., VINCENDON, G., ET GOMBOS, G., Une m6thode densitom6trique de d6termination quantitative de la fraction prot6ique S-100, Ann. Biol. clin., in press. 19 UYEMURA, K., TARDY, J., VINCENDON, G., MANDEL, P., ET GOMBOS, G., Mise en 6vidence de prot6ines sp6cifiques du cerveau chez les Mammif6res, C.R. Soc. Biol. (Paris), 161 (1967) 1396-1399. 20 WENDELL-SMITH,C. P., BLUNT, M. J., BALDWIN, F., AND BAISLEY,P. B., Neurone-satellite cell relationship, Nature (Lond.), 205 (1965) 781-782. 21 ZUCI~ERMAN,J, E., HERSCHMAN,J. R., AND LEVINE, L., Appearance of a brain specific antigen (the S-100 protein) during human foetal development, J. Neurochem., 17 (1970) 247-251. (Accepted Decemberl8th, 1970)
Brain Research, 26 (1971) 475-479
Erratum Brain Research, 26 (1971) 177-183 T h e m a g n i f i c a t i o n s o f F i g u r e s 1, 2 a n d 4 read ' × 'x
100';
t h e y should read
20, x 23 a n d x 18', r e s p e c t i v e l y .
Brain Research, 26 (1971) 479