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Regional distribution of superoxide dismutase in rat brain during postnatal development
Developmental Brain Research, 4 (1982) 333-337
333
Elsevier Biomedical Press
Regional Distribution of Superoxide Dismutase in Rat Brain During Postnatal Development MARC LEDIG, RAINER FRIED, MARTINE ZIESSEL and PAUL MANDEL Centre de Neurochimie du CNRS, 5, Rue Blaise Pascal, 67084 Strasbourg Cddex (France) and (R.F.) Department of Biochemistry, Creighton University Medical School, Omaha, NE 68178 (U.S.A.)
(Accepted December 3rd, 1981) Key words: superoxide dismutase - - brain - - brain regions - - brain development - - catecholamines
Superoxide dismutase in nervous system protects readily oxidizable compounds such as catecholamines against toxic effects of oxygen. We investigated superoxide dismutase activity during development in 5 brain regions selected for a wide range of catecholamine concentration and turnover: cerebellum, neocortex, striatum, hypothalamus and medulla-pons. The cytosolic and the particulate enzyme were measured from birth to 6 months of age. In cerebellum the cytosolic enzyme shows considerable activity on the first postnatal days; the particulate enzyme is less active, both reach a maximum at 3 months. In cortex and striatum both activities were low during the postnatal days and reach a plateau at 3 months. In hypothalamus both activities are higher during the postnatal days and reach a maximum at 3 months. In medulla-pons the values are 2 times higher than in all other regions; the cytosolic enzyme reaches a maximum at 2 months whereas the particulate enzyme reaches a plateau at 3 months. Thus our results show an increase of superoxide dismutase activity during development in all brain regions; the highest activities were found in regions with high catecholamine content. INTRODUCTION The brain is an organ which has both a high oxygen consumption as well as a high concentration of unsaturated lipids and catecholamines and other compounds which can be readily oxidized. Part of the system of antioxidant defenses is the enzyme superoxide dismutase (EC 1.15.1.1) which is present in rather high amounts, distributed throughout the brain and in most subcellular fractions (see review in ref. 6). This enzyme has been studied extensively; however, a systematic study of its activity in different brain regions and at diverse developmental stages has not yet been carried out. This becomes the more important since the biochemical composition of discrete anatomical brain regions show considerable variations, both in enzyme activities and neurotransmitter content which can present a different rhythm during developmentZ,a,10, especially in cerebellum (for review see ref. 35). Superoxide dismutase in the nervous system presumably protects readily oxidizable compounds like 0165-3806/82/0000-0000/$02.75 © Elsevier Biomedical Press
catecholamines and subcellular zones, such as membranes, against the toxic effects of oxygen 6. The present research paper deals with a study of superoxide dismutase activity during development in 5 brain regions, selected for a wide range of catecholamine concentration and turnoverg,12,32: cerebellum, neocortex, medulla-pons, hypothalamus, and striatum. The subcellular distribution of enzyme is apt to vary during developmentL The bulk of superoxide dismutase is located in the cytosolic fractions, and represents the Cu, Zn-enzyme; all studied subcellular fractions show enzyme activity16,30,a1. Particulate superoxide dismutase is reported to be a mixture of the Mn-enzyme and the Cu, Zn-enzymelS,27,a2. It is generally assumed that the Cu, Zn-enzyme is inhibited by 1 m M K C N , while the Mn-enzyme is not inhibited24,aa; however, several investigators report a requirement of up to 10 m M to inhibit the Cu, Znenzyme completely (refs. 4, 26, Oberley, Iowa City, personal communication). The validity of this assumption is tested in the present paper.
334 MATERIALS AND METHODS
Male Wistar rats from the colony of the Centre de Neurochimie were used. They had been kept on stock rat pellet diet receiving food and water ad libitum. All animals were killed by stunning and decapitation. Individual brains were removed immediately after killing, rinsed with iced saline, and chilled on an iced glassplate for dissection. Major brain regions were dissected, using a guiding device previously described 19. Anatomical regions were immediately placed in liquid nitrogen and stored at --70 °C till assay. The dissected brain regions were thawed and homogenized at about 2 °C in 5 vol. 0.9 ~ NaC1 in a Potter-Elvehjem glass-homogenizer with Teflon pestle, and were centrifuged at 100,000 g at 4 °C for 1 h. The supernatant was dialyzed overnight at 4 °C against 3 changes of 0.1 M sodium-phosphate, pH 7.8, and were used without further purification for assay of superoxide dismutase. This fraction corresponds to the cytosolic isoenzyme. The original precipitate after centrifugation was suspended in 5 vols. of 0.9 ~o NaC1, and frozen and thawed, 3 times between 37 °C and liquid nitrogen, and centrifuged 1 h at 100,000 g. The resulting supernatant was dialyzed and assayed without further purification for particulate superoxide dismutase. Brain portions from animals of one week or older were processed individually (n = 8), brain regions from smaller animals were pooled. Superoxide dismutase was assayed colorimetrically s by the NADH-nitro-blue tetrazolium assay at 540 nm at 25 °C, as previously described 14. Bovine erythrocyte superoxide dismutase was used as reference. Activity is expressed as #g SOD/mg protein. Cyanide was used to inhibit the Cu, Zn-enzyme 4,26, 32. Proteins were determined colorimetricallyis with bovine serum albumin as standard. All assays were carried out in duplicate. Nitro-blue tetrazolium (grade III) phenazine methosulfate, bovine erythrocyte superoxide dismutase and N A D H were obtained from Sigma (St. Louis). All other reagents were reagent grade.
solic isoenzyme shows considerable activity on the first postnatal days. Cytosolic and particulate activity reach a maximum in about 90 days; the activity of the particulate enzyme rises more sharply during the first month. Both activities decrease in adult rats, which is in agreement with previous reports dealing with SOD activity in the whole brain 13,21,as. A sharp rise in Mn superoxide dismutase during the first two months has been reported z3. Superoxide activity in 4 other anatomical brain regions was assayed (Fig. 2). Great variations are found both in level as well as in the age-related changes of superoxide dismutase activity. Cytosolic enzyme activity increases from birth to a plateau at 2 months for neocortex and striatum, and to a peak at 2-3 months for hypothalamus and medulla-pons. High activity is found in the hypothalamus and specially in the medulla-pons. In general, the cytosolic isoenzyme and the particulate isoenzymes (all expressed in terms of Cu, Zn-superoxide dismutase) show parallel changes in activity during development.
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RESULTS AND DISCUSSION
Superoxide dismutase was assayed in rat cerebellum from birth to 6 months of age (Fig. 1). Cyto-
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SUPEROXIDE DISHUTASE OF DEVELOPING BRAIN A : PARTICULATE OF CEREBELLUH ( A ) S : CYTOSOLIC OF CEREBELLUH (/~)
Fig. I.
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SUPEROXIDE DISMUTASE OF DEVELOPINGBRAIN, A : PARTICULATE S : CYTOSOLIC NEOCORTEX ( 0 ) , STRIATUI,I ([-1), HYPOTHALAMUS((~), MEDULLA-PONS ( ~ ) ,
Fig. 2. The complexity of changes of enzyme activity during development have been discussed by Van den Berg 3. It should be noted that these changes may have a different pattern in different anatomical regions of:~he nervous system, and even the intracellular distribution of the enzyme among subcellular fractions may vary. Oxygen consumption itself increases considerably during maturation of the brain; it is, therefore, not surprising also to find variations of enzymes related to oxygen metabolism. Superoxide dismutase activity had been studied in the whole brain of developing rats~l,22,2a. It is interesting to note that while brain enzymes increase sharply during the first 2-3 months, enzymes in lung and erythrocytes show very slight variations2L High activity had been found in 1-day-old rats in brain, lung and retinas of rats s. A sharp rise in superoxide dismutase activity was reported in brains of rats and
mice during the first 2 monthslL While there is general agreement in the findings reported by different authors; the data, unfortunately, are not readily comparable in detail, due to different assay methods used, as well as different dissection of anatomical brain regions. Superoxide dismutase during prenatal development has not yet been studied systematically. High activity was found in whole chick embryo aged 15 and 20 days, (M. Ledig, unpublished results). The embryonic liver of two inbred strains of mice shows a sharply rising SOD activity between age 10 and 20; it is interesting to note that both the total levels throughout the lifespan of the animals, as well as the changes in SOD activity vary greatly among the two strains ~5. Superoxide dismutase prevents the oxidative inactivation of catecholamine and other compounds as well as oxidation damage to membranes and other intracellular structures. The distribution and turnover of catecholamines varies greatly among different anatomical brain regionsg,13,zL The hypothalamus shows the highest concentration of catecholamines, and the slowest turnover rate; on the other hand, low steady-state levels of catecholamines and high turnover is found in the cerebellum. Thus, in hypothalamus catecholamines persist for longer periods than in cerebellum. One would expect higher superoxide activity in regions where oxidation of the aromatic ring should be prevented. The distribution of superoxide dismutase was found generally to parallel high concentrations of catecholamines in different brain regions16, al. Enzymatic oxidation of the aromatic ring of catecholamines has been documented in mammalian brain1,11,17,25. Superoxide dismutase would protect catecholamines against this oxidation by scavenging the oxygen species required for this reaction. The distribution of superoxide dismutase has been examined in major brain regions. Since there seems to be a parallelism between catecholamine accumulations and superoxide dismutase, it would be worthwhile to extend these studies to small regions such as nuclei, where catecholamine metabolism is particularly intense. It is also of interest to note here that the synaptosomal regions show a much higher superoxide dismutase activity than the mitochondrial fractions 31.
336 TABLE I Effect of cyanide on superoxide dismutase in cerebellum of rats Assay
Cytosol Activity*
% inhibition
Control (1 month) 1 mM KCN 5 mM KCN
9.10 (10.6-7.6) 5.70 (3.5-7.7) 1.85 (1.3-3.2)
37 79
Control (5-6 months) 1 mM 5 mM KCN
7.50 (4.8-11.9) 2.57 (2.0- 3.4) 0.41 (0.00-0.7)
63 95
* ttg SOD/mg protein (n ~- 6). C y a n i d e was used to discriminate between the M n - e n z y m e a n d the Cu, Z n - e n z y m e ; we f o u n d 1 m M K C N to be insufficient to inhibit the cytosolic enzyme completely, which was o b t a i n e d only with 5 m M K C N (Table I). This confirms the findings o f Oberley a n d colleagues4, ~ . L 6 n n e r d a l a n d colleagues 15 f o u n d one isoenzyme in rat a n d mouse brain, insensitive to 20 m M cyanide, and 5 a n d 3 isoenzymes respectively inhibited by cyanide, as well as n u m e r o u s isoenzymes with different isoelectric points in liver. One should c o m m e n t here on a c u s t o m prevailing in the litera-
ture, where lack o f inhibition by cyanide per se is considered to indicate the presence o f the M n enzyme it w o u l d be worthwhile to confirm this by actual t r a c e - m e t a l analysis. Conversely, M n - e n z y m e is frequently given as difference between ( ' t o t a l ' 'cyanide-sensitive') superoxide dismutase, again using 1 m M cyanide as discriminating inhibitor. The most valid designation would be 'cyanide-sensitive' a n d 'cyanide-insensitive' enzyme fractions, while metal content a n d intracellular distribution should not be c o n c l u d e d just from inhibition by low concentrations o f cyanide. Thus, 'cyanide-insensitive' superoxide dismutase has been r e p o r t e d in m a m m a lian o r g a n s : h u m a n a n d chicken liver, as well as h u m a n a n d chicken heart, a n d mouse liver, brain, heart a n d lung, as well as in extracts o f m a m m a l i a n brain by electrophoresis15,20,24,27,29,33, 34. Partially purified superoxide dismutase has been studied in m a m m a l i a n brainT, 8. C o m p l e t e purification o f either isoenzyme from m a m m a l i a n b r a i n has not yet been reported. ACKNOWLEDGEMENTS W e should like to t h a n k M. Serge G o b a i l l e for skillful dissection o f brains.
REFERENCES 1 Barras, B. C., Coult, D. B., Rich, P. and Tutt, K. J., Substrate specificity of caeruloplasmin. Phenylalkylamine substrates, Biochem. Pharmacol., 23 (1974) 45-56. 2 Benjamins, J. A. and McKhann, G. M., Development, regeneration, and aging. In G. J. Siegel, R. W. Albers, R. Katzman and B. W. Agranoff (Eds.), Basic Neurochemistry, 2nd Edn., Little Brown, Boston, 1976, pp. 365-387. 3 Van den Berg, C. J., Enzymes in the developing brain. In W. Himwich (Ed.), Biochemistry of the Developing Brain, Marcel Dekker, New York, 1974, pp. 149-198. 4 Bize, I. B., Oberley, L. W. and Morris, H. P., Superoxide dismutase and superoxide radical in Morris hepatomas, Cancer Res., 40 (1980) 3686-3692. 5 Fried, R., Enzymatic and non-enzymatic assay of superoxide dismutase, Biochimie, 57 (1975) 657-660. 6 Fried, R., Superoxide dismutase activity in the nervous system, J. Neurosci. Res., 4 (1979) 435-441. 7 Fried, R., Fried, L. W. and Babin, D. R., The inhibition of reduction of tetrazolium by a liver protein, Europ. J. Biochem., 16 (1970) 399-406. 8 Fried, R. and Mandel, P., Superoxide dismutase of mammalian nervous system, J. Neurochem., 24 (1975) 433-438. 9 Glowinski, J. and Iversen, L. L., Regional studies of catecholamines in the rat brain. I. The disposition of
10 11 12
13 14
15 16
dopamine and DOPA in various regions of the brain, J. Neurochem., 13 (1966) 655-669. Himwieh, W. (Ed.), Biochemistry of the Developing Brain, Vols. 1 and 2, Marcel Dekker, New York, 1973/1974. lnchiosa, M. A., Jr. and Rodrigui~z~I. B., Distribution of an epinephrine-oxidizing enzyme in mammalian tissues, Biochem. Pharmacol., 18 (1969) 2032~2935. Iversen, L. L. and Glowinski, J., Regional studies of catecholamines in the rat brain. II. Rate of turnover of catecholamines in various brain regions, J. Neurochem., 13 (1966) 671-682. Kellogg, E. W., III and Fridovich, I, Superoxide dismutase in the rat and mouse as a function of age and longevity, J. Gerontol., 31 (1976) 405-408. Ledig, M., M'Paria, J. M., Louis, J. C., Fried, R. and Mandel, P., Effect of ethanol on superoxide dismutase activity in cultured neural cells, Neurochem. Res., 5 (1980) 1155-1162. Lbnnerdal, B., Keen, C. L. and Hurley, L. S., Isoelectric focusing of superoxide dismutase isoenzymes, FEBS Lett., 108 (1979) 51-55. Loomis, T. C., Yeh, G. and Stahl, W. L., Regional and subcellular distribution of superoxide dismutase in brain, Experientia, 32 (1976) 1374-1376.
337 17 Lovstad, R. A., Effect of centrally active drugs on dopamine oxidation by rat brain catecholamine oxidase, Experientia, 35 (1979) 1642-1645. 18 Lowry, O. H., Rosebrough, N. FI., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 19 Mack, G., Contribution a l'Etude des Bases Moldculaires du Comportement Muricide chez le Rat, Thesis, Universit6 Louis Pasteur, Strasbourg, France, 1978. 20 Marklund, S., Purification and characterization of a manganese containing superoxide dismutase from bovine heart mitochondria, Int. J. Biochem., 9 (1978) 299-306. 21 Matkovics, B., Effect of plant and animal tissue lesions of superoxide dismutase activities. In A. M. Michelson, J. M. McCord and I. Fridovich (Eds.), Superoxide and Superoxide Dismutases, Academic Press, New York, 1977, pp. 501-515. 22 Mavelli, I., Mondovi, B., Federico, R. and Rotilio, G., Superoxide dismutase activity in developing rat brain, J. Neurochem., 31 (1978) 363-364. 23 Mavelli, I. and Federico, R., Superoxide dismutase, catalase and glutathione peroxidase activity in developing rat brain, ItaL J. Biochem., 29 (1980) 48-50. 24 McCord, J. M., Boyle, J. A., Day, E. D., Jr., Rizzolo, L. J. and Salin, M. L., A manganese-containingsuperoxide dismutase from human liver. In A. M. Michelson, J. M. McCord and I. Fridovich (Eds.), Superoxide and Superoxide Dismutase from Human Liver, Academic Press, New York, 1977, pp. 129-138. 25 Novak, R., Matkovics, B., Marik, M. and Fachet, J., Changes in mouse liver superoxide dismutase activity and lipid peroxidation during embryonic and postpartum development, Experientia, 34 (1978) 1134-1135. 26 Oberley, L. W., Bize, I. B., Sahu, S. K., Leuthauser, S. W.
27 28 29 30 31 32
33 34 35 36
H. C. and Gruber, H. E., Superoxide dismutase activity of normal murine liver, regenerating liver and H6 hepatoma, J. nat. Cancer Inst., 61 (1978) 375-379. Peeters-Joris, C., Vandevorde, A. M. and Baudhuin, P., Subcellular localization of superoxide dismutase in rat liver, Biochem. J., 150 (1975) 31-39. Prohaska, J. R. and Wells, W. W., Copper deficiency in the developing rat brain: a possible model for Menkes' steely-hair disease, J. Neurochem., 23 (1974) 91-98. deRosa, G., Keen, C. L., Leach, R. M. and Hurley, L. S., Regulation of superoxide dismutase activity by dietary manganese, J. Nutr., 110 (1980) 795-804. Savolainen, H., Superoxide dismutase and glutathione peroxidase activities in rat brain, Res. Commun. Chem. Pathol. Pharmacol., 21 (1978) 173-176. Thomas, T. N., Priest, D. G. and Zemp, J. W., Distribution of superoxide dismutase in rat brain, J. Neurochem., 27 0976) 309-310. Versteeg, D. H. G., van der Gugten, J., de Jong, W. and Palkovits, M., Regional concentration of noradrenaline and dopamine in rat brain, Brain Res., 113 (1976) 563574. Weisiger, R. A. and Fridovich, I., Superoxide dismutase. Organelle specificity, J. biol. Chem., 248 (1973) 35823592. Weisiger, R. A. and Fridovich, I., Mitochondrial superoxide dismutase site of synthesis and intramitochondrial localization, J. biol. Chem., 248 (1973) 4793-4796. Van der Wende, C. and Spoerlein, M. T., Oxidation of dopamine to melanin by an enzyme of rat brain, Life Sci., 2 (1963) 386-392. Zanetta, J. P., Federico, A. and Vincendon, G., Glycosidases and cerebellar ontogenesis in the rat, J. Neurochem., 34 (1980) 831-834.
333
Elsevier Biomedical Press
Regional Distribution of Superoxide Dismutase in Rat Brain During Postnatal Development MARC LEDIG, RAINER FRIED, MARTINE ZIESSEL and PAUL MANDEL Centre de Neurochimie du CNRS, 5, Rue Blaise Pascal, 67084 Strasbourg Cddex (France) and (R.F.) Department of Biochemistry, Creighton University Medical School, Omaha, NE 68178 (U.S.A.)
(Accepted December 3rd, 1981) Key words: superoxide dismutase - - brain - - brain regions - - brain development - - catecholamines
Superoxide dismutase in nervous system protects readily oxidizable compounds such as catecholamines against toxic effects of oxygen. We investigated superoxide dismutase activity during development in 5 brain regions selected for a wide range of catecholamine concentration and turnover: cerebellum, neocortex, striatum, hypothalamus and medulla-pons. The cytosolic and the particulate enzyme were measured from birth to 6 months of age. In cerebellum the cytosolic enzyme shows considerable activity on the first postnatal days; the particulate enzyme is less active, both reach a maximum at 3 months. In cortex and striatum both activities were low during the postnatal days and reach a plateau at 3 months. In hypothalamus both activities are higher during the postnatal days and reach a maximum at 3 months. In medulla-pons the values are 2 times higher than in all other regions; the cytosolic enzyme reaches a maximum at 2 months whereas the particulate enzyme reaches a plateau at 3 months. Thus our results show an increase of superoxide dismutase activity during development in all brain regions; the highest activities were found in regions with high catecholamine content. INTRODUCTION The brain is an organ which has both a high oxygen consumption as well as a high concentration of unsaturated lipids and catecholamines and other compounds which can be readily oxidized. Part of the system of antioxidant defenses is the enzyme superoxide dismutase (EC 1.15.1.1) which is present in rather high amounts, distributed throughout the brain and in most subcellular fractions (see review in ref. 6). This enzyme has been studied extensively; however, a systematic study of its activity in different brain regions and at diverse developmental stages has not yet been carried out. This becomes the more important since the biochemical composition of discrete anatomical brain regions show considerable variations, both in enzyme activities and neurotransmitter content which can present a different rhythm during developmentZ,a,10, especially in cerebellum (for review see ref. 35). Superoxide dismutase in the nervous system presumably protects readily oxidizable compounds like 0165-3806/82/0000-0000/$02.75 © Elsevier Biomedical Press
catecholamines and subcellular zones, such as membranes, against the toxic effects of oxygen 6. The present research paper deals with a study of superoxide dismutase activity during development in 5 brain regions, selected for a wide range of catecholamine concentration and turnoverg,12,32: cerebellum, neocortex, medulla-pons, hypothalamus, and striatum. The subcellular distribution of enzyme is apt to vary during developmentL The bulk of superoxide dismutase is located in the cytosolic fractions, and represents the Cu, Zn-enzyme; all studied subcellular fractions show enzyme activity16,30,a1. Particulate superoxide dismutase is reported to be a mixture of the Mn-enzyme and the Cu, Zn-enzymelS,27,a2. It is generally assumed that the Cu, Zn-enzyme is inhibited by 1 m M K C N , while the Mn-enzyme is not inhibited24,aa; however, several investigators report a requirement of up to 10 m M to inhibit the Cu, Znenzyme completely (refs. 4, 26, Oberley, Iowa City, personal communication). The validity of this assumption is tested in the present paper.
334 MATERIALS AND METHODS
Male Wistar rats from the colony of the Centre de Neurochimie were used. They had been kept on stock rat pellet diet receiving food and water ad libitum. All animals were killed by stunning and decapitation. Individual brains were removed immediately after killing, rinsed with iced saline, and chilled on an iced glassplate for dissection. Major brain regions were dissected, using a guiding device previously described 19. Anatomical regions were immediately placed in liquid nitrogen and stored at --70 °C till assay. The dissected brain regions were thawed and homogenized at about 2 °C in 5 vol. 0.9 ~ NaC1 in a Potter-Elvehjem glass-homogenizer with Teflon pestle, and were centrifuged at 100,000 g at 4 °C for 1 h. The supernatant was dialyzed overnight at 4 °C against 3 changes of 0.1 M sodium-phosphate, pH 7.8, and were used without further purification for assay of superoxide dismutase. This fraction corresponds to the cytosolic isoenzyme. The original precipitate after centrifugation was suspended in 5 vols. of 0.9 ~o NaC1, and frozen and thawed, 3 times between 37 °C and liquid nitrogen, and centrifuged 1 h at 100,000 g. The resulting supernatant was dialyzed and assayed without further purification for particulate superoxide dismutase. Brain portions from animals of one week or older were processed individually (n = 8), brain regions from smaller animals were pooled. Superoxide dismutase was assayed colorimetrically s by the NADH-nitro-blue tetrazolium assay at 540 nm at 25 °C, as previously described 14. Bovine erythrocyte superoxide dismutase was used as reference. Activity is expressed as #g SOD/mg protein. Cyanide was used to inhibit the Cu, Zn-enzyme 4,26, 32. Proteins were determined colorimetricallyis with bovine serum albumin as standard. All assays were carried out in duplicate. Nitro-blue tetrazolium (grade III) phenazine methosulfate, bovine erythrocyte superoxide dismutase and N A D H were obtained from Sigma (St. Louis). All other reagents were reagent grade.
solic isoenzyme shows considerable activity on the first postnatal days. Cytosolic and particulate activity reach a maximum in about 90 days; the activity of the particulate enzyme rises more sharply during the first month. Both activities decrease in adult rats, which is in agreement with previous reports dealing with SOD activity in the whole brain 13,21,as. A sharp rise in Mn superoxide dismutase during the first two months has been reported z3. Superoxide activity in 4 other anatomical brain regions was assayed (Fig. 2). Great variations are found both in level as well as in the age-related changes of superoxide dismutase activity. Cytosolic enzyme activity increases from birth to a plateau at 2 months for neocortex and striatum, and to a peak at 2-3 months for hypothalamus and medulla-pons. High activity is found in the hypothalamus and specially in the medulla-pons. In general, the cytosolic isoenzyme and the particulate isoenzymes (all expressed in terms of Cu, Zn-superoxide dismutase) show parallel changes in activity during development.
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RESULTS AND DISCUSSION
Superoxide dismutase was assayed in rat cerebellum from birth to 6 months of age (Fig. 1). Cyto-
months
months
SUPEROXIDE DISHUTASE OF DEVELOPING BRAIN A : PARTICULATE OF CEREBELLUH ( A ) S : CYTOSOLIC OF CEREBELLUH (/~)
Fig. I.
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SUPEROXIDE DISMUTASE OF DEVELOPINGBRAIN, A : PARTICULATE S : CYTOSOLIC NEOCORTEX ( 0 ) , STRIATUI,I ([-1), HYPOTHALAMUS((~), MEDULLA-PONS ( ~ ) ,
Fig. 2. The complexity of changes of enzyme activity during development have been discussed by Van den Berg 3. It should be noted that these changes may have a different pattern in different anatomical regions of:~he nervous system, and even the intracellular distribution of the enzyme among subcellular fractions may vary. Oxygen consumption itself increases considerably during maturation of the brain; it is, therefore, not surprising also to find variations of enzymes related to oxygen metabolism. Superoxide dismutase activity had been studied in the whole brain of developing rats~l,22,2a. It is interesting to note that while brain enzymes increase sharply during the first 2-3 months, enzymes in lung and erythrocytes show very slight variations2L High activity had been found in 1-day-old rats in brain, lung and retinas of rats s. A sharp rise in superoxide dismutase activity was reported in brains of rats and
mice during the first 2 monthslL While there is general agreement in the findings reported by different authors; the data, unfortunately, are not readily comparable in detail, due to different assay methods used, as well as different dissection of anatomical brain regions. Superoxide dismutase during prenatal development has not yet been studied systematically. High activity was found in whole chick embryo aged 15 and 20 days, (M. Ledig, unpublished results). The embryonic liver of two inbred strains of mice shows a sharply rising SOD activity between age 10 and 20; it is interesting to note that both the total levels throughout the lifespan of the animals, as well as the changes in SOD activity vary greatly among the two strains ~5. Superoxide dismutase prevents the oxidative inactivation of catecholamine and other compounds as well as oxidation damage to membranes and other intracellular structures. The distribution and turnover of catecholamines varies greatly among different anatomical brain regionsg,13,zL The hypothalamus shows the highest concentration of catecholamines, and the slowest turnover rate; on the other hand, low steady-state levels of catecholamines and high turnover is found in the cerebellum. Thus, in hypothalamus catecholamines persist for longer periods than in cerebellum. One would expect higher superoxide activity in regions where oxidation of the aromatic ring should be prevented. The distribution of superoxide dismutase was found generally to parallel high concentrations of catecholamines in different brain regions16, al. Enzymatic oxidation of the aromatic ring of catecholamines has been documented in mammalian brain1,11,17,25. Superoxide dismutase would protect catecholamines against this oxidation by scavenging the oxygen species required for this reaction. The distribution of superoxide dismutase has been examined in major brain regions. Since there seems to be a parallelism between catecholamine accumulations and superoxide dismutase, it would be worthwhile to extend these studies to small regions such as nuclei, where catecholamine metabolism is particularly intense. It is also of interest to note here that the synaptosomal regions show a much higher superoxide dismutase activity than the mitochondrial fractions 31.
336 TABLE I Effect of cyanide on superoxide dismutase in cerebellum of rats Assay
Cytosol Activity*
% inhibition
Control (1 month) 1 mM KCN 5 mM KCN
9.10 (10.6-7.6) 5.70 (3.5-7.7) 1.85 (1.3-3.2)
37 79
Control (5-6 months) 1 mM 5 mM KCN
7.50 (4.8-11.9) 2.57 (2.0- 3.4) 0.41 (0.00-0.7)
63 95
* ttg SOD/mg protein (n ~- 6). C y a n i d e was used to discriminate between the M n - e n z y m e a n d the Cu, Z n - e n z y m e ; we f o u n d 1 m M K C N to be insufficient to inhibit the cytosolic enzyme completely, which was o b t a i n e d only with 5 m M K C N (Table I). This confirms the findings o f Oberley a n d colleagues4, ~ . L 6 n n e r d a l a n d colleagues 15 f o u n d one isoenzyme in rat a n d mouse brain, insensitive to 20 m M cyanide, and 5 a n d 3 isoenzymes respectively inhibited by cyanide, as well as n u m e r o u s isoenzymes with different isoelectric points in liver. One should c o m m e n t here on a c u s t o m prevailing in the litera-
ture, where lack o f inhibition by cyanide per se is considered to indicate the presence o f the M n enzyme it w o u l d be worthwhile to confirm this by actual t r a c e - m e t a l analysis. Conversely, M n - e n z y m e is frequently given as difference between ( ' t o t a l ' 'cyanide-sensitive') superoxide dismutase, again using 1 m M cyanide as discriminating inhibitor. The most valid designation would be 'cyanide-sensitive' a n d 'cyanide-insensitive' enzyme fractions, while metal content a n d intracellular distribution should not be c o n c l u d e d just from inhibition by low concentrations o f cyanide. Thus, 'cyanide-insensitive' superoxide dismutase has been r e p o r t e d in m a m m a lian o r g a n s : h u m a n a n d chicken liver, as well as h u m a n a n d chicken heart, a n d mouse liver, brain, heart a n d lung, as well as in extracts o f m a m m a l i a n brain by electrophoresis15,20,24,27,29,33, 34. Partially purified superoxide dismutase has been studied in m a m m a l i a n brainT, 8. C o m p l e t e purification o f either isoenzyme from m a m m a l i a n b r a i n has not yet been reported. ACKNOWLEDGEMENTS W e should like to t h a n k M. Serge G o b a i l l e for skillful dissection o f brains.
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