Journal
OJ
Nrurocheniistry, 1975.
Vol. 24. pp. 433-438. Pergamon Press. Printed in Great Britain.
SUPEROXIDE DISMUTASE OF MAMMALIAN NERVOUS SYSTEM R. FRIED’and P. MANDEL Centre de Neurochimie du CNRS, 11 Rue Humann, 67085 Strasbourg Cedex, France (Received 13 June 1974. Accepted 29 July 1974)
Abstract-Superoxide dismutase was assayed in portions of the nervous system (a) by inhibition of tetrazolium reduction by oxygen radicals, generated enzymically, and (b) by inhibition of tetrazolium reduction by oxygen radicals generated by oxidation of NADH in presence of phenazine methosulphate. Superoxide dismutase activity was found in beef brain, retina and adrenal medulla, as well as in brain, retina and lungs of adult and of newborn rats. Preliminary experiments with rats exposed to hyperbaric oxygen showed no alteration of enzyme activities in tissues of newborn and adult animals. The possible role of superoxide dismutase in the nervous system is discussed.
CEREBROCUPREIN, a metallo-protein, was described in nervous system would lead to a better understanding mammalian and human brain by PORTER & FOLCH of the role and mechanism of this enzyme. Retina and (1957) about 20 y ago; later it was found to be identi- adrenal medulla present two readily accessible porcal with or closely related to similar ‘cupreins’ from tions of the nervous system which show high oxidative & DEUTSCH, 1969). The super- activity, and in which enzymes involved in oxygen other tissues (CARRICO oxide dismutase activity of these proteins was estab- metabolism, such as superoxide dismutase, would be lished by M C C ~ R& D FRIDOVICH (1969). Meanwhile, expected to play a prominent role. Both of these tissues superoxide dismutase, mainly prepared from liver and have been extensively used as models for biochemistry erythrocytes, has been intensely studied (FRIDOVICH,of the nervous system in this laboratory (MANDEL et 1972;WESER, 1973). Superoxide dismutase of mamma- al., 1974; AUNISet al., 1973). The present study deals lian brain was studied in our laboratory, and had been with the superoxide dismutase from beef and rat brain originallycalled ‘tetrazoliumreductase inhibitor’(FRIED as well as that of bovine retina and adrenal medulla. & FRIED,1967; FRIEDet al., 1970, 1973); the enzyme Preliminary data is also presented on the effect of & hyperbaric oxygen on selected tissues of newborn rats, is also identical with ‘tetrazolium oxidase’ (LIPPIT FRIDOVICH, 1973). No essential differences in enzymic since activity of this enzyme might be altered when activity and properties were found in superoxide dis- high oxygen concentrations are present. The enzyme is mutase preparations from mammalian liver and brain assayed by inhibition of tetrazolium reduction due to (FRIED & FRIED, 1967; FRIED et al., 1970, 1973). While oxygen radicals, generated either by an enzymic system xanthine oxidase) (FRIEDet al., 1970), superoxide dismutase acts as a universal protecting (xanthine agent against singlet oxygen and oxygen radicals (FRI- 1973) or by a new nonenzymic method (FRED,1974) DOVICH, 1972; POLITZER et al., 1971; WESER, 1973), it based on findings of NISHIKIMI et al. (1972). is not known as yet, whether it has a special function in the nervous system. MATERIALS AND METHODS Previous studies on superoxide dismutase activity of Superoxide dismutase was prepared from organs the CNS had been carried out with preparations obtained from whole brain (FRIEDet al., 1970, 1973). obtained from freshly slaughtered animals. Beef brain, adrenals and whole eyeballs were packed in ice and proSince this presents a very complex system, it was cessed within 1 h of slaughter. Male adult, or newborn Wisthought that a study of a more uniform portion of the tar rats were used for studies of rat tissues. Retinas were iso-
+
lated according to KLETHI ei 01. (1970); subcellular fractions On leave from the Department of Biochemistry, Creigh- of bovine adrenal medulla were obtained by centrifugation ton University, Medical School, Omaha (NB 68178, USA.). in sucrose gradients (AUNISet al., 1973)and were kindly proAbbreuiatior~used: HPO, hyperbaric oxygen. vided by AUNIS& MRAS-PORTUGAL. Superoxide dismutase 433
R. FRIEDand P. MANDEL
434
was prepared as previously described (FRIEDet a/., 1970, 1973) by acid precipitation and ammonium sulphate fractionation followed-for more highly purified brain enzyme -by heat and chloroform treatment. The effect of hyperbaric oxygen was tested with Wistar rats, through the kind co-operation of Dr. MANTZ(Service de Rtanimation, HBpital Civil, Strasbourg). Animals were kept for I h in the oxygenation tank with 100% oxygen at 3 atm absolute (MANTZ& TEMPE,1968). For determination of enzyme activity in the retina of newborn rats, the scalp was peeled off, eyes were excised. placed in iced phosphate buffer 0.1 M, pH 7.8 (‘phosphate’)and dissected. Cornea, lens and intraocular fluid were removed, and the remaining portion, containing the sclera, was designated ‘retina’. Groups of newborn rats were used with a total of 160 animals; for each experiment 15-20 rats were oxygenated and their tissues pooled. These preparations were compared with those from an equal number of control rats not subjected to oxygen treatment and processed at the same time and by the same methods. Organs were homogenized in a PotterElvehjem tissue homogenizer, and dialysed 3 times for 30 rnin against phosphate, before enzyme determination. Partial purification of the superoxide dismutase was done with undialysed homogenates, using the simplified standard procedure, up to ammonium sulphate fractionation. Xanthine oxidase (cream xanthine oxidase, Sigma) and superoxide dismutase were assayed as previously described (FRIED,1966; FRIED& FRIED,1970; FRIEDet al., 1970, 1973) by aerobic reduction of Nitro-Blue tetrazolium, at 25°C. pH 7.8, at 540 nm in an Yvon-Jobin spectrophotometer. For purified enzyme or fractions with high superoxide dismutase activity, xanthine oxidase was used to give about 1.0 absorbance change at 540 nm per 10 min (‘high’); for fractions or preparations with low dismutase activity, xanthine oxidase controls were set at about 0 3 absorbance units/30 min (‘low’). For comparative studies of superoxide dismutase, this enzyme was added to give about 50 per cent inhibition. In addition to generating the oxygen radicals enzymically by xanthine oxidase, they were also formed in a non-enzymic system (FRIED,1974),based on the aerobic oxidation of NADH by phenazine methosulphate in presence of Nitroet a/., 1972). Superoxide dismuBlue tetrazolium (NISHIKIMI vase activity was determined as a proportion of tetrazolium reduced in presence and absence of this enzyme. Protein concentration was determined by the method of LOWRYet al. (1951).Fine chemicals were obtained from Sigma (U.S.A.) and Boehringer (Germany). All other reagents were Reagent Grade. RESULTS
Superoxide dismutase activity was found in dialysed homogenate from whole brain; activity of this enzyme was inversely related to the amount of xanthine oxidase added, in agreement with previous findings (FRIED e t a / . , 1970, 1973; FRIED, 1974; Tables I and 2). Animals killed by ether anaesthesia showed somewhat higher activity levels than those killed without ether, especially in the assays with partially purified enzyme.
A more highly purified fraction of beef brain superoxide dismutase showed greater specific activity (FRIED, 1974). Preliminary evidence indicates that a specific inhibitor or masking factor of superoxide dismutase is removed during this process. Work is currently in progress to clarify this point. In addition to whole brain, superoxide dismutase was also assayed in other tissues related to the nervous system, either directly in the dialysed homogenate or in a partially purified fraction. Superoxide dismutase activity was found in homogenates of retina from beef and adult rats, and was further purified from beef retina, using the same method as we habitually apply for liver and brain preparations. Superoxide dismutase prepared from beef retina has approximately the same level of activity as that from brain; graded levels of enzyme cause increasing inhibition of the xanthine dehydrogenase assay system, and superoxide dismutase found is inversely related to the amount of xanthine oxidase added (Fig. 1). The retina as well as selected other tissues from newborn rats were also tested for superoxide dismutase activity, prepared by an abbreviated routine method. Activity is already present in tissues of newborn rats, and does not vary greatly between these animals and adults (Fig. 2 and Table 2). Taurine has been proposed as a neurotransmitter in the retina, where it is found in high concentration (MANDELet al., 1974). Taurine has also been correclated with regulation of oxygen metabolism, and is said to counteract the inhibition of oxygen uptake due to cyanide (SCUTERI,1970). Since superoxide dismutase, an enzyme Flosely related to oxygen metabolism, is also inhibited by cyanide (FRIED et a/., 1970 1973), a possible correlation between taurine metabolism and superoxide dismutase in the retina was looked for. Taurine was tested as possible substrate or inhibitor of xanthine oxidase (in the absence of superoxide dismutase) over a wide concentration range and wide proportion range in relation to hypoxanthine and did not affect xanthine oxidase activity under any condition. Taurine had likewise no effect on purified superoxide dismutase prepared either from beef retina or brain, and did not reverse the inhibition of superoxide dismutase by cyanide. The antagonistic effect between taurine and cyanide in tissue respiration, discussed by SCUTERI (1970) does, therefore, not involve oxygen radicals o r superoxide dismutase (FRIED,1973, unpublished). Beef adrenal medulla was separated into subcellular fractions, which were tested for superoxide dismutase activity directly or after a shortened purification method based on the method routinely used in our laboratory. No superoxide dismutase activity was found in chromaffin granules, or other particulate fractions, but high activity was found in the supernatant
Superoxide dismutase
TABLEI . per assay
102 115
(control) 108 92
(control) 200 200
(control) 190 150
OF RAT BRAIN
A/30 min
o
A. Brain homogenate 0.30 2 018 9 0.18
-
8 12 -
7 7
40 40
-
-
0.19 0.19 0.29 0.15 0.15
31 37
~
50 50
-
-
-
10 7
0.17 0.17
43 43
B. Partially purified enzyme -
0.83 0.8 1 0.76 0.93 0.86 0.80 060 0.4 1 0.38 0.70 0.48 0.56
(control) 85 92
(control) 50 88
(control) 110 82
(control) 50 54
-
0.22 009 0.07
2 9 -
-
-
-
31 37
0.4 1 0.36 0.34 0.4 1 0.39 0.35 0.41 0.3 1 0.30 0.4 1 0.32 0.34
61 70
0.1 10 0.06 0.15 0.03 0.0 1
8 8
NADH oxidation A 540 Inhib.
% Inhib.
-
0.83 0.8 1 0.76 0.93 0.86 0.82 0.49 0.46 0.46 0.56 0.50 0.53
(control)
DISMUTASE
‘Low x
‘High XO’ AjlO min % Inhib.
pg Protein
Rat
SUPEROXIDE
435
50 75
80 90
-
-
-
31 20
0.03 0.03
80 80
~
~
-
12 18 -
6 16
24 28
23 17
~~
Eight male adult Wistar rats (250-300 g) were killed by stunning (Nos. 1-4) or light ether anaesthesia (Nos. 5-8), and decapitation. Rat brains and partial purifications of superoxide dismutase were processed individually. Homogenates of whole brains were dialysed 3 x 30 min at 4°C against 0.1 M-Na-phosphate, pH 7.8. Superoxide dismutase was partially purified from whole brain hornogenates by acid precipitation and ammonium sulphate fractionation. All assays were carried out in duplicate, at 25”C, pH 7.8, in 0.1 M-phosphate buffer. Absorbance was read at 540 nm.XO, xanthine oxidase.
fraction. As was reported above for other tissues, superoxide dismutase activity in the supernatant fraction of adrenal medulla was directly related to the amount of enzyme added (Fig. 3). This intracellular distribution is in agreement with that reported for liver
preparations (WEISIGER & FRIDOVICH, 1973; ROTILIOet al., 1973).The presence of superoxide dismutase in the soluble fraction is in agreement with a recent paper (PETRACK &CHER~K 1974) , that tyrosine hydroxylase, another enzyme found in the soluble fraction of
TABLE 2. EFFECT OF OXYGENATIONON SUPEROXIDE IXSMUTASE Organ
HPO
OF ORGANS FROM ADULT RATS
pg Protein in assay
‘Low XO*’
% Inhib.
NADH*
% Inhib.
-
042 028 0.27 0-34 0.3 1
-
270 250 96 72 36 32
0.175 003 0.03 0.07 0.04 011 0.08
~~
Control Lung Brain Retina
~~
-
+ ++
81 81 59 73 31 53
040 0.37
33 36 19 27 6 12
*Freshly prepared fraction was used for the xanthine oxidase assay; the same preparation was used for NADH assays after being stored at 4°C for about 10 weeks. Six adult Wistar rats were kept at 3 atm of 100% oxygen (HPO) at 25°C for 2 h. They were killed by ether anaesthesia; superoxide dismutase was partially purified from organ homogenates. Rats not receiving oxygen treatment served as controls. XO, xanthine oxidase.
436
R. FRIED and P. MANDEL
A540
XO=O.lml O t
05
0 4
02
02
01
I70
r
10
20
3
20
10
30
5
min
FIG. 1. Superoxide dismutase purified from beef retina. The enzyme was assayed by 'xanthine oxidaselow' method. Numbers along curves indicate concentration of superoxide dismutase, in pg of protein/3.0 ml; 30 rnin at 2 5 T , pH 7.8. Graded levels of superoxide dismutase were tested with two concentrations OF xanthine oxidase.
adrenal medulla, does not involve the oxygen radical mechanism, and, indeed, is inhibited by .O;. Superoxide dismutase is considered to be an enzyme which protects cells and sensitive molecules against the deleterious effects of oxygen radicals or singlet oxygen (FRIDOVICH, 1972; FRIED et al., 1973; LAVELLE et al.,
'I 90
.O
1973). Hyperbaric oxygen (HPO) is gaining increasing importance as therapeutic agent for several types of respiratory diseases and intoxications (MANTZ & TEMPE,1968) and is also important for orbital flights, as well as emergency treatments for premature babies. Toxic effects are frequently observed, with the retina,
d
A U
0
I
I
I00
200
I 300
I 400
1 500 '1300
150
OJ
Nrurocheniistry, 1975.
Vol. 24. pp. 433-438. Pergamon Press. Printed in Great Britain.
SUPEROXIDE DISMUTASE OF MAMMALIAN NERVOUS SYSTEM R. FRIED’and P. MANDEL Centre de Neurochimie du CNRS, 11 Rue Humann, 67085 Strasbourg Cedex, France (Received 13 June 1974. Accepted 29 July 1974)
Abstract-Superoxide dismutase was assayed in portions of the nervous system (a) by inhibition of tetrazolium reduction by oxygen radicals, generated enzymically, and (b) by inhibition of tetrazolium reduction by oxygen radicals generated by oxidation of NADH in presence of phenazine methosulphate. Superoxide dismutase activity was found in beef brain, retina and adrenal medulla, as well as in brain, retina and lungs of adult and of newborn rats. Preliminary experiments with rats exposed to hyperbaric oxygen showed no alteration of enzyme activities in tissues of newborn and adult animals. The possible role of superoxide dismutase in the nervous system is discussed.
CEREBROCUPREIN, a metallo-protein, was described in nervous system would lead to a better understanding mammalian and human brain by PORTER & FOLCH of the role and mechanism of this enzyme. Retina and (1957) about 20 y ago; later it was found to be identi- adrenal medulla present two readily accessible porcal with or closely related to similar ‘cupreins’ from tions of the nervous system which show high oxidative & DEUTSCH, 1969). The super- activity, and in which enzymes involved in oxygen other tissues (CARRICO oxide dismutase activity of these proteins was estab- metabolism, such as superoxide dismutase, would be lished by M C C ~ R& D FRIDOVICH (1969). Meanwhile, expected to play a prominent role. Both of these tissues superoxide dismutase, mainly prepared from liver and have been extensively used as models for biochemistry erythrocytes, has been intensely studied (FRIDOVICH,of the nervous system in this laboratory (MANDEL et 1972;WESER, 1973). Superoxide dismutase of mamma- al., 1974; AUNISet al., 1973). The present study deals lian brain was studied in our laboratory, and had been with the superoxide dismutase from beef and rat brain originallycalled ‘tetrazoliumreductase inhibitor’(FRIED as well as that of bovine retina and adrenal medulla. & FRIED,1967; FRIEDet al., 1970, 1973); the enzyme Preliminary data is also presented on the effect of & hyperbaric oxygen on selected tissues of newborn rats, is also identical with ‘tetrazolium oxidase’ (LIPPIT FRIDOVICH, 1973). No essential differences in enzymic since activity of this enzyme might be altered when activity and properties were found in superoxide dis- high oxygen concentrations are present. The enzyme is mutase preparations from mammalian liver and brain assayed by inhibition of tetrazolium reduction due to (FRIED & FRIED, 1967; FRIED et al., 1970, 1973). While oxygen radicals, generated either by an enzymic system xanthine oxidase) (FRIEDet al., 1970), superoxide dismutase acts as a universal protecting (xanthine agent against singlet oxygen and oxygen radicals (FRI- 1973) or by a new nonenzymic method (FRED,1974) DOVICH, 1972; POLITZER et al., 1971; WESER, 1973), it based on findings of NISHIKIMI et al. (1972). is not known as yet, whether it has a special function in the nervous system. MATERIALS AND METHODS Previous studies on superoxide dismutase activity of Superoxide dismutase was prepared from organs the CNS had been carried out with preparations obtained from whole brain (FRIEDet al., 1970, 1973). obtained from freshly slaughtered animals. Beef brain, adrenals and whole eyeballs were packed in ice and proSince this presents a very complex system, it was cessed within 1 h of slaughter. Male adult, or newborn Wisthought that a study of a more uniform portion of the tar rats were used for studies of rat tissues. Retinas were iso-
+
lated according to KLETHI ei 01. (1970); subcellular fractions On leave from the Department of Biochemistry, Creigh- of bovine adrenal medulla were obtained by centrifugation ton University, Medical School, Omaha (NB 68178, USA.). in sucrose gradients (AUNISet al., 1973)and were kindly proAbbreuiatior~used: HPO, hyperbaric oxygen. vided by AUNIS& MRAS-PORTUGAL. Superoxide dismutase 433
R. FRIEDand P. MANDEL
434
was prepared as previously described (FRIEDet a/., 1970, 1973) by acid precipitation and ammonium sulphate fractionation followed-for more highly purified brain enzyme -by heat and chloroform treatment. The effect of hyperbaric oxygen was tested with Wistar rats, through the kind co-operation of Dr. MANTZ(Service de Rtanimation, HBpital Civil, Strasbourg). Animals were kept for I h in the oxygenation tank with 100% oxygen at 3 atm absolute (MANTZ& TEMPE,1968). For determination of enzyme activity in the retina of newborn rats, the scalp was peeled off, eyes were excised. placed in iced phosphate buffer 0.1 M, pH 7.8 (‘phosphate’)and dissected. Cornea, lens and intraocular fluid were removed, and the remaining portion, containing the sclera, was designated ‘retina’. Groups of newborn rats were used with a total of 160 animals; for each experiment 15-20 rats were oxygenated and their tissues pooled. These preparations were compared with those from an equal number of control rats not subjected to oxygen treatment and processed at the same time and by the same methods. Organs were homogenized in a PotterElvehjem tissue homogenizer, and dialysed 3 times for 30 rnin against phosphate, before enzyme determination. Partial purification of the superoxide dismutase was done with undialysed homogenates, using the simplified standard procedure, up to ammonium sulphate fractionation. Xanthine oxidase (cream xanthine oxidase, Sigma) and superoxide dismutase were assayed as previously described (FRIED,1966; FRIED& FRIED,1970; FRIEDet al., 1970, 1973) by aerobic reduction of Nitro-Blue tetrazolium, at 25°C. pH 7.8, at 540 nm in an Yvon-Jobin spectrophotometer. For purified enzyme or fractions with high superoxide dismutase activity, xanthine oxidase was used to give about 1.0 absorbance change at 540 nm per 10 min (‘high’); for fractions or preparations with low dismutase activity, xanthine oxidase controls were set at about 0 3 absorbance units/30 min (‘low’). For comparative studies of superoxide dismutase, this enzyme was added to give about 50 per cent inhibition. In addition to generating the oxygen radicals enzymically by xanthine oxidase, they were also formed in a non-enzymic system (FRIED,1974),based on the aerobic oxidation of NADH by phenazine methosulphate in presence of Nitroet a/., 1972). Superoxide dismuBlue tetrazolium (NISHIKIMI vase activity was determined as a proportion of tetrazolium reduced in presence and absence of this enzyme. Protein concentration was determined by the method of LOWRYet al. (1951).Fine chemicals were obtained from Sigma (U.S.A.) and Boehringer (Germany). All other reagents were Reagent Grade. RESULTS
Superoxide dismutase activity was found in dialysed homogenate from whole brain; activity of this enzyme was inversely related to the amount of xanthine oxidase added, in agreement with previous findings (FRIED e t a / . , 1970, 1973; FRIED, 1974; Tables I and 2). Animals killed by ether anaesthesia showed somewhat higher activity levels than those killed without ether, especially in the assays with partially purified enzyme.
A more highly purified fraction of beef brain superoxide dismutase showed greater specific activity (FRIED, 1974). Preliminary evidence indicates that a specific inhibitor or masking factor of superoxide dismutase is removed during this process. Work is currently in progress to clarify this point. In addition to whole brain, superoxide dismutase was also assayed in other tissues related to the nervous system, either directly in the dialysed homogenate or in a partially purified fraction. Superoxide dismutase activity was found in homogenates of retina from beef and adult rats, and was further purified from beef retina, using the same method as we habitually apply for liver and brain preparations. Superoxide dismutase prepared from beef retina has approximately the same level of activity as that from brain; graded levels of enzyme cause increasing inhibition of the xanthine dehydrogenase assay system, and superoxide dismutase found is inversely related to the amount of xanthine oxidase added (Fig. 1). The retina as well as selected other tissues from newborn rats were also tested for superoxide dismutase activity, prepared by an abbreviated routine method. Activity is already present in tissues of newborn rats, and does not vary greatly between these animals and adults (Fig. 2 and Table 2). Taurine has been proposed as a neurotransmitter in the retina, where it is found in high concentration (MANDELet al., 1974). Taurine has also been correclated with regulation of oxygen metabolism, and is said to counteract the inhibition of oxygen uptake due to cyanide (SCUTERI,1970). Since superoxide dismutase, an enzyme Flosely related to oxygen metabolism, is also inhibited by cyanide (FRIED et a/., 1970 1973), a possible correlation between taurine metabolism and superoxide dismutase in the retina was looked for. Taurine was tested as possible substrate or inhibitor of xanthine oxidase (in the absence of superoxide dismutase) over a wide concentration range and wide proportion range in relation to hypoxanthine and did not affect xanthine oxidase activity under any condition. Taurine had likewise no effect on purified superoxide dismutase prepared either from beef retina or brain, and did not reverse the inhibition of superoxide dismutase by cyanide. The antagonistic effect between taurine and cyanide in tissue respiration, discussed by SCUTERI (1970) does, therefore, not involve oxygen radicals o r superoxide dismutase (FRIED,1973, unpublished). Beef adrenal medulla was separated into subcellular fractions, which were tested for superoxide dismutase activity directly or after a shortened purification method based on the method routinely used in our laboratory. No superoxide dismutase activity was found in chromaffin granules, or other particulate fractions, but high activity was found in the supernatant
Superoxide dismutase
TABLEI . per assay
102 115
(control) 108 92
(control) 200 200
(control) 190 150
OF RAT BRAIN
A/30 min
o
A. Brain homogenate 0.30 2 018 9 0.18
-
8 12 -
7 7
40 40
-
-
0.19 0.19 0.29 0.15 0.15
31 37
~
50 50
-
-
-
10 7
0.17 0.17
43 43
B. Partially purified enzyme -
0.83 0.8 1 0.76 0.93 0.86 0.80 060 0.4 1 0.38 0.70 0.48 0.56
(control) 85 92
(control) 50 88
(control) 110 82
(control) 50 54
-
0.22 009 0.07
2 9 -
-
-
-
31 37
0.4 1 0.36 0.34 0.4 1 0.39 0.35 0.41 0.3 1 0.30 0.4 1 0.32 0.34
61 70
0.1 10 0.06 0.15 0.03 0.0 1
8 8
NADH oxidation A 540 Inhib.
% Inhib.
-
0.83 0.8 1 0.76 0.93 0.86 0.82 0.49 0.46 0.46 0.56 0.50 0.53
(control)
DISMUTASE
‘Low x
‘High XO’ AjlO min % Inhib.
pg Protein
Rat
SUPEROXIDE
435
50 75
80 90
-
-
-
31 20
0.03 0.03
80 80
~
~
-
12 18 -
6 16
24 28
23 17
~~
Eight male adult Wistar rats (250-300 g) were killed by stunning (Nos. 1-4) or light ether anaesthesia (Nos. 5-8), and decapitation. Rat brains and partial purifications of superoxide dismutase were processed individually. Homogenates of whole brains were dialysed 3 x 30 min at 4°C against 0.1 M-Na-phosphate, pH 7.8. Superoxide dismutase was partially purified from whole brain hornogenates by acid precipitation and ammonium sulphate fractionation. All assays were carried out in duplicate, at 25”C, pH 7.8, in 0.1 M-phosphate buffer. Absorbance was read at 540 nm.XO, xanthine oxidase.
fraction. As was reported above for other tissues, superoxide dismutase activity in the supernatant fraction of adrenal medulla was directly related to the amount of enzyme added (Fig. 3). This intracellular distribution is in agreement with that reported for liver
preparations (WEISIGER & FRIDOVICH, 1973; ROTILIOet al., 1973).The presence of superoxide dismutase in the soluble fraction is in agreement with a recent paper (PETRACK &CHER~K 1974) , that tyrosine hydroxylase, another enzyme found in the soluble fraction of
TABLE 2. EFFECT OF OXYGENATIONON SUPEROXIDE IXSMUTASE Organ
HPO
OF ORGANS FROM ADULT RATS
pg Protein in assay
‘Low XO*’
% Inhib.
NADH*
% Inhib.
-
042 028 0.27 0-34 0.3 1
-
270 250 96 72 36 32
0.175 003 0.03 0.07 0.04 011 0.08
~~
Control Lung Brain Retina
~~
-
+ ++
81 81 59 73 31 53
040 0.37
33 36 19 27 6 12
*Freshly prepared fraction was used for the xanthine oxidase assay; the same preparation was used for NADH assays after being stored at 4°C for about 10 weeks. Six adult Wistar rats were kept at 3 atm of 100% oxygen (HPO) at 25°C for 2 h. They were killed by ether anaesthesia; superoxide dismutase was partially purified from organ homogenates. Rats not receiving oxygen treatment served as controls. XO, xanthine oxidase.
436
R. FRIED and P. MANDEL
A540
XO=O.lml O t
05
0 4
02
02
01
I70
r
10
20
3
20
10
30
5
min
FIG. 1. Superoxide dismutase purified from beef retina. The enzyme was assayed by 'xanthine oxidaselow' method. Numbers along curves indicate concentration of superoxide dismutase, in pg of protein/3.0 ml; 30 rnin at 2 5 T , pH 7.8. Graded levels of superoxide dismutase were tested with two concentrations OF xanthine oxidase.
adrenal medulla, does not involve the oxygen radical mechanism, and, indeed, is inhibited by .O;. Superoxide dismutase is considered to be an enzyme which protects cells and sensitive molecules against the deleterious effects of oxygen radicals or singlet oxygen (FRIDOVICH, 1972; FRIED et al., 1973; LAVELLE et al.,
'I 90
.O
1973). Hyperbaric oxygen (HPO) is gaining increasing importance as therapeutic agent for several types of respiratory diseases and intoxications (MANTZ & TEMPE,1968) and is also important for orbital flights, as well as emergency treatments for premature babies. Toxic effects are frequently observed, with the retina,
d
A U
0
I
I
I00
200
I 300
I 400
1 500 '1300
150
