Comp. Biochem. Physiol., 1977, Vol. 56B, pp. 397 to 402. Pergamon Press. Printed in Great Britain
A COMPARATIVE STUDY OF SOME MORE IMPORTANT EXPERIMENTAL ANIMAL PEROXIDE METABOLISM ENZYMES* B. MATKOVICSt,R. NOV/,K, HOANG DUC HANI-I,L. SZAB6 AND SZ. I. VARGA Biological Isotope Laboratory, Institute of Biochemistry, "A.J." University of Szeged, Hungary
(Received 26 July 1976) Abstraet--t. We have studied and compared the peroxide metabolism enzymes (SOD, P and C) of the main organs of fresh-water mollusc, chicken, mouse, guinea-pig, rabbit, cat and dog. 2. The liver exhibited the highest SOD activity. The enzymatic activities of the organ homogenates of the guinea-pig stand out in comparison with the values for the other homogenates examined. 3. The liver, kidney and total brain homogenates of the chicken and the vertebrates display no, or only a very 10w P activity. The highest P activities were measured in the haemolysates. 4. The C and SOD values exhibit a certain parallelism. 5. The peroxide metabolism enzyme activities calculated by utilizing the protein measurements permit the establishment of a more realistic enzymatic activity. INTRODUCTION In an earlier publication (Matkovics et al., 1976) quantitative data measured in organ and tissue homogenates were reported for SOD (EC 1.15.1.1.). This enzyme catalyzes the dismutation of 0 2 anions formed from molecular oxygen in biological systems: O2- + 0 2 + 2 H + - " ) H 2 0 2 + 02
(a)
SOD therefore gives rise to H202, in the decomposition of which the other two enzymes, studied also by us, contained iron-porphyrin skeleton, P (EC 1.11.1.7.) and C (EC 1.11.1.6.), take part. P is able to react with H202 only in the presence of proton donors (b), while the reaction catalyzed by C resembles the over described dismutation reaction
(c): H202 + AH2 ~ 2 H 2 0 + A
(b)
H202 + H202 c__}2 H 2 0 + 02
(C)
Although not only SOD produces H202 in tissues and organs, nevertheless the comprehensive term "peroxide metabolism enzymes" is a fitting one for the enzymes we have studied and described, for they can be intercorrelated in an evolutionary context (Frieden, 1974), and P and C are among the most active enzymes of H202 decomposition; however, it is true that they are not the sole decomposers of H202 in the living world.
The tissues were prepared, and the enzymatic activities were measured and calculated, as described in detail earlier (Matkovics et al., 1976; Part VIII/A). The centrifuged supernatants of the homogenates were used for every enzyme and other measurement. Homogenization was performed in a Potter glass homogenizer, with ice-cooling. SOD was measured quantitatively at pH 10.2, 25°C and 480 nm by the adrenochrome method of Misra & Fridovich (1972). P activities were determined at 25°C and 470 nm by the spectrophotometric guaiacol method of George (1955) (In the book of Methods in Enzymology). The method of Beers & Sizer (1952), involving backmeasurement of the rate of H202 consumption at 240 nm and 25°C, was used for quantitative determination of C activities. Enzymatic activity units were defined previously too (see Part VIII/A). Quantitative protein measurements were made by the method of Lowry et al. (1955), based on the Folin-phenol reagent, bovine plasma albumin being used to prepare the standard curve at 25°C and 675 nm. The reagents employed in enzymatic activity determinations were products of Reanal (Budapest, Hungary), Merck (Darmstadt, GFR) and Calbiochem (Basel, Switzerland) of the highest purity. They could thus be used without preliminary purification. For visible and ultraviolet spectrophotometric measurements, Spektromom 360, Spektromom 2"131(both MOM, Budapest, Hungary) and VSU-2P (Zeiss, Jena, GDR) spectrophotometers were utilized.
MATERIALS AND METHODS
RESULTS AND DISCUSSION
The experimental objects investigated were as follows: fresh-water shell-fish (Anodonta cyfnea), chicken (Gallus domesticus), mouse (Mus musculus, Strain CBA-H2K male), guinea-pig (Carla cutleri), rabbit (Oryctolaxjus cuniculus), cat (Felis catus) and dog (Canis familiaris).
The tissue and organ SOD, P and C values of the experimental animals investigated earlier (the snail, frog, carp, pigeon, rat and pig) were supplemented with peroxide metabolism enzyme measurements on a further 7 animals used mainly also for physiological and pharmacological experimental purposes. These results are given in the tables. It should be noted in general that the tabulated data are the averages of at least 10 measurements. The mean error of the measurements is _4- 5-10~o too.
*Part VIII/B of the series "Properties of enzymes". f To whom reprint requests and correspondence should be addressed (P.O. Box 539, H-6701 Szeged, Hungary). Abbreviations used: as previously and as follows: freshwater shell-fish = f.w.sh.f. (mollusc); guinea-pig = g.p. 397
308
B. MATKOVICS et al. Table 1. Organs kidney liver spleen whole brain lung pancreas thymus f ovarium genital ~ uterus organs ~ testes stomach or chicken gizzard intestine colon • f.w.sh.f. muscle muscles heart muscle skeletal ,muscle )" hemolysate blood l, plasma gills f.w.sh.f, genital organs
f.w.sh.f,
chicken
mouse
715
1060 3370 360 350 300 350
1470 4450 415 565 340 380
120 238 285
g.p. rabbit 1228 5600 635 635 470 630 104 715 635 395
cat
dog
375 3500 500 380 200 300
1200 1760 2950 3370 100 560 300 799 190 560 180 475 395 100 900 96 320 100
176 352 336
300 290
310 272 144
0 650
425
795
500
560
635
300 300 1
280 600 24
256 790 24
305 1500
300 900
313 1600 6
0 715
SOD activities of various animal organ and tissue homogenates, measured at 480 nm and 25°C, and calculated as enzyme units (U) per g wet tissue. In the calculation of the data, a PL/1 p r o g r a m m e was used on a R H O (Robotron, Magdeburg, G D R ) computer. (The problem of computer solution of enzymatic activity calculation will be dealt with in a separate paper in the near future.) Tables 1-3 contain enzymatic activities of only the main tissues and organs, and some endocrine glands.
Because of the very interesting nature of the results obtained to date, problems relating to the peroxide metabolism activities of the endocrine glands in particular will be dealt with in a separate publication. These experimental data suggest that it will be possible to draw certain endocrine evolutionary conclusions.
Table 2. Organs kidney liver spleen whole brain lung pancreas thymus ( ovarium genital ~ uterus organs t, testes stomach or chicken gizzard intestine colon ( f.w.sh.f. | muscle heart muscles } muscle / skeletal I muscle ~ hemolysate blood plasma gills f.w.sh.f, genital organs
f.w.sh.f,
chicken
mouse
2200
0 0 1100 0 2900 30
0 0 2500 0 1900 145 450 500
1000 60
g.p. rabbit 0 0 620 120 1350 60
20 10 800 85 600 75
150 270 300
180 155
cat
dog
180 10 1220 110 1000 120 180
0 0 700 130 1300 90 200 210
400
30 150 90
160 200
20 160 100
750
570
710
90 7500 360 300
82 ---
90
40 70
1600
720
86 20950 280
100 9500 100
90
2400 0
P activities of various animal organ and tissue homogenates (U)g wet tissue.
450
Animal peroxide metabolism enzymes
399
Table 3. Organs kidney liver spleen whole brain lung pancreas thymus genital )" ovarium uterus organs ~ testes stomach or chicken gizzard intestine colon r f.w.sh.f. | muscle muscles ~ heart | muscle | skeletal 1. muscle hemolysate blood plasma gills f.w.sh.f, genital organs
f.w.sh.f,
chicken
0.220
0.265 2.010 0.260 0.020 0.119 0.320
mouse
g.p. rabbit cat
dog
9.600 4.800 2.400 0.320 8.055 10.500 2.140 2.200 3.340 1.458 1.175 1.580 1.600 0.660 1.500 0.070 1.104 0.040 0.060 0.100 0.195 1.860 0.480 0.598 2.000 0.210 0.960 0.200 0.120 1.965 0.800 0.582 0.940 0.240 - 1.000 0.840 0.120 0.940 0.205 0.104 0.123
0.142 0.060
---
0.153
0.195
1.660 - 0.700 - 0.420
-0.695 0.400 0.500 0.410 0.240
0.009
0.100 4.020 0.0
0.580 0.230 0.324 0.600
0.090 0.244 0.070 0.152 0.256 90.000 11.400 0.600 - - 11.555 0.040 0.182 0.030 - 0.195
0.082 0.100
C activities of various animal organ and tissue homogenates (B.U./g wet tissue). Table 1 lists the SOD values. For the mollusc, only the branchia, kidney, plantaris and stomach data are given. In place of the stomach data, those of the gizzard are listed for the chicken. (Our data relating to human tissues up to now from normal biopsies are only few to be given in a separate column.) Analysis of the data in Table 1 leads to the following conclusions regarding the SOD values of the organs and tissues examined: (a) As in the earlier results (Part VIII/& 1976) here too the liver exhibits the highest SOD activities. The guinea-pig liver has an outstandingly high SOD activity, while the values for the dog, chicken and rabbit livers are very similar to one another. (b) In most cases the kidney homogenates have SOD activities about 3 times lower than those of the liver; in some cases (chicken and dog), these activities approach those of the hemolysates, which are all of nearly the same magnitude. The blood plasma exhibits no, or only a very low activity of SOD (this may perhaps be correlated with the slight spontaneous hemolysis of the red blood cells). (c) The SOD activities of the other tissue and organ homogenates lie in the range 50-800 U/g wet tissue. Similarly, conclusions may be drawn from the quantitative P results in Table 2: (a) The highest P activities in the red blood cell hemolysates were found in the chicken, mouse and rabbit, the chicken exhibiting the largest values. No P activity was observed in the red blood cells of the dog and guinea-pig. The plasma P activities lie in the range 100450 U/g. (b) The highest P activities are found in the organs of the chicken, while the P occurs to the greatest extent in the tissue and organ homogenates of the g.p., cat and dog.
(c) There is in general no, or only low P activity in the liver and kidney tissues. The kidney and branchia of the mollusc exhibit high P activities. (d) The activity sequence for the various tissue and organ homogenates is as follows: hemolysate > lung > spleen > myocardium. Table 3 gives the C activities in B.U./g wet tissue. Here too, several points may be noted: (a) The highest C activities are those of the hemolysates, that of the mouse blood hemolysate being outstanding at 90.0 B.U./g wet tissue. (b) The most complete values are those relating to the organ and tissue homogenates of the guinea-pig. (c) The C activity sequence is: hemolysate > liver > kidney > spleen > lung. (d) The C activities of the other organ and tissue homogenates lie in the range 0.009-1.00 B.U./g wet tissue, the only exception being that of the homogenate of the g.p. ovary (1.660 B.U./g wet tissue). (e) The C values for all of the brain tissue homogenates studied are markedly low. (We shall deal with this problem separately in the description of the specific activities.) If the C activities of the organ and tissue homogenates are made the subject of a general study, then it can be seen at once that the C activities provide a picture of the aerobic metabolism of the tissues. More exactly, the C activity values are higher in those tissues more strongly connected with the aerobic metabolism and with molecular oxygen, or carrying out active protein synthesis. In Table 4 we wish to put our studies to date in a new light. This table contains our measurements on tissue proteins reported by Lowry et al. (1951);
400
B. MATKOV1CSet al. Table 4. Organs kidney liver spleen whole brain lung pancreas genital ~" ovarium uterus organs ( testes stomach or chicken gizzard intestine colon • f.w.sh.f. muscle heart muscles muscle skeletal muscle blood hemolysate • plasma gills f.w.sh.f, genital organs
f.w.sh.f,
chicken
mouse
g.p.
dog
50
50 150 70 36 70 78
142.8 196 93.2 48 88 91
56 140 67 42 63 77 46 32 45
70 90 50 43 65 72 50 40
36 41 37
40 43 35
45
46
57.2 47 45 20 45
124
45 120* 150"
58.4 130* 110"
40
45
110"
100"
130"
128'
16 38
Protein contents of various animal organ and tissue homogenates (mg protein/g wet tissue)• these are later used to present the specific activities of the peroxide metabolism enzymes studied. In our protein determinations the results are given in mg protein/g wet tissue. Protein values were determined for organ and tissue homogenates from mollusc, mouse, chicken, g.p. and dog. In the following tables we shall see the specific activities of the peroxide metabolism enzymes for the animals listed in Table 4, in enzyme units/mg protein. Table 5 gives the specific enzymatic activities for the mollusc. In this and the subsequent tables the specific activity sequence will be SOD, P and C. In the mollusc organ examinations, no special effort was made to prepare the ganglia, for example, but the specific activities of the peroxide metabolism enzymes were investigated on only four easily preparable organs. The tables are interesting in that there are modifications in the sequences of enzymatic activities relating to the organ and tissue homogenates as established earlier in connection with the specific enzymatic activities; this is due to the increase in the Table 5. Organs kidney genital organs muscles gills
SOD
P
C
14.3
44
0.0044
18.8 0 0.0030 0 2.0 0.0004 0 150 0.0050
Specific peroxide metabolism enzymatic activities of mollusc organ and tissue homogenates (enzyme U/mg protein).
value of the denominator in the quotient. For instance, in Table 5 the specific S O D activities of the reproductive system precede the values measured for the kidney homogenates. The specific P and C activities are highest of all for the mollusc branchia. Table 6 lists the specific enzymatic activities relating to the mouse tissues. It is interesting to observe that the brain lies in third place, after the liver and kidney, in the sequence of S O D specific activities, standing out from the moderate S O D activities exhibited by the other organ and tissue homogenates, which barely differ from one another. The mouse brain, liver and kidney do not exhibit P activity. The high specific P activity of the hemolysate is followed directly by that of the spleen. Table 6. Organs
SOD
P
C
kidney liver spleen whole brain lung pancreas testes
12.09 22.47 4.64 12.05 3.85 3.19 1.085
0 0 31.0 0 21.1 1.67 9.24
0.072 0.055 0.0129 0.015 0.002 0.0023 0.0033
4.25
17.7
0.002
4.83 4.6 0.2
1.58 73.2 0.9
0.0012 0.6923 0.0003
muscles blood
[" heart ,) muscle ] skeletal t muscle hemolysate plasma
Specific peroxide metabolism enzymatic activities of mouse organ and tissue homogenates (U/mg protein).
Animal peroxide metabolism enzymes
401 Table 9.
Table 7. Organs
SOD
P
C
kidney liver spleen whole brain lung pancreas intestine
21.2 22.5 5.1 9.7 4.3 4.5 6.3
0.0 0.0 15.7 0.0 41.4 0.4 1.3
0.005 0.013 0.004 0.001 0.002 0.004 0.001
14.4
1.6
0.003
1.9 139.7 2.3 21.3
0.002 0.026 0.0 0.003
muscles blood
heart muscle skeletal muscle hemolysate plasma
gizzard
6.6 2.0 0.008 5.1
Specific peroxide metabolism enzymatic activities of chicken organ and tissue homogenates (U/mg protein). In comparison with the outstanding specific C activity of the hemolysate, the mouse liver and kidney have moderate activities, and the other homogenates low values. Table 7 presents the specific peroxide metabolism enzyme values for the organ and tissue homogenates of the chicken. As regards the SOD specific enzymatic activities, those of the liver, kidney and myocardium (in decreasing order) exceed 10 U/mg protein. Of the P specific activities, here too the outstanding value for the hemolysate is followed by high values for the lung, gizzard and spleen. C exhibits high activities in the hemolysate and liver, and low activities in the other homogenates examined. In Table 8 we find the specific activities of the peroxide metabolism enzymes in the organs and tissues of the guinea-pig. In order of magnitude, SOD activities of more than 10U/mg protein are exhibited by the liver, the adrenal, the kidney, the myocardium, the uterus, the ovary and the brain. The SOD activity of the liver homogenate here is 40.0 U/mg protein. Table 8. Organs
SOD
P
C
kidney liver spleen whole brain lung pancreas thymus genital )- ovarium uterus organs ~ testes stomach intestine colon hemolysate blood plasma
21.9 40.0 9.5 15.1 7.5 8.2 4.5 15.5 19.8 8.8 4.9 8.6 9.1 6.1 0.2
0.0 0.0 9.3 2.9 21.4 0.8 19.6 3.2 8.4 6.7 0.8 3.7 2.4 0.0 3.3
0.0857 0.0153 0.0236 0.0025 0.0295 0.0124 0.0347 0.0204 0.0263 0.0023 0.0461 0.0171 0.0114 0.0877 0.0017
Specific peroxide metabolism enzymatic activities of guinea-pig organ and tissue homogenates (U/mg protein). C.B.P. 56/4B--D
Organs
SOD
P
C
kidney liver spleen whole brain lung pancreas genital J" ovarium organs ~ uterus stomach intestine colon heart muscle muscles skeletal muscle hemolysate blood plasma
25.1 37.4 11.2 18.6 8.6 6.6 18.0 8.0 7.8 6.3 4.1
0.0 0.0 14.0 3.0 20.0 1.3 4.0 5.3 0.5 3.7 2.9
0.1151 0.0162 0.0300 0.0023 0.0307 0.0273 0.0200 0.0235 0.0173 0.0116 0.0069
13.8
15.4
0.0130
7.0 12.5 0.06
3.0 0.0 4.5
0.0057 0.0903 0.0020
Specific peroxide metabolism enzymatic activities of dog organ and tissue homogenates (U/rag protein). The sequence for the higher P specific activities is headed by the lung and the thymus, with the myocardium and spleen in third and fourth places. The kidney, adrenal, liver and hemolysate do not exhibit P activity. The C values in Table 8 lead to the following findings: (a) The C activity of the adrenal generally exceeds the highest C activity of the hemolysate, which is also approached by that of the kidney homogenate. (b) The other measured C activities lie in the range 0.0017-O.0461 B.U./mg protein. Table 9 lists the specific activities of the peroxide metabolism enzymes in the organs and tissues of the dog. As regards the SOD activities, the highest is that of the liver here too, followed by that of the adrenal. The P specific activity sequence is the lung, the myocardium and the spleen. The highest specific C activity is found in the adrenal. To give a brief summary of all the above data, it may be said, mainly from a comparison of the results relating to the enzyme U/g wet tissue and the specific enzyme U/mg protein values, that the enzymatic activities obtained via quantitative protein determinations provide a new picture in the comparison of the peroxide metabolism enzymes. Both sets of data have their own value. The more extreme values of the enzymatic activities calculated per g of wet tissue are rather conspicuous, whereas the specific activities are more realistic.
REFERENCES
BEERSR. F. JR. • SIZER I. W. (1952) Spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase, d. biol. Chem. 195, 133-140. COLOWICKS. P. & KAPLAr~N. O. (1955) Methods in Enzymology, Vol. 2. pp. 764-775. Academic Press, New York. FRmOEN E. (1974) Biochemical evolution of iron and copper proteins. Chem. & Eng. News. March 25, pp. 42-46.
402
B. MATKOV1CSet al.
LOWRY O. H., ROSEBROUGHN. J., FARR A. L. RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MATKOVICS B., NOV/~K R., HOANG DUC HANH, SZAB6 L., VARGA Sz. I. • ZALESNAG. (1976) A comparative study of some more important experimental animal peroxide
metabolism enzymes. (VIII/A) Comp. Biochem. Physiol. (In press.) MISRA H. P. & FRIDOVICH I. (1972) The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J. biol. Chem. 247, 3170-3175.
A COMPARATIVE STUDY OF SOME MORE IMPORTANT EXPERIMENTAL ANIMAL PEROXIDE METABOLISM ENZYMES* B. MATKOVICSt,R. NOV/,K, HOANG DUC HANI-I,L. SZAB6 AND SZ. I. VARGA Biological Isotope Laboratory, Institute of Biochemistry, "A.J." University of Szeged, Hungary
(Received 26 July 1976) Abstraet--t. We have studied and compared the peroxide metabolism enzymes (SOD, P and C) of the main organs of fresh-water mollusc, chicken, mouse, guinea-pig, rabbit, cat and dog. 2. The liver exhibited the highest SOD activity. The enzymatic activities of the organ homogenates of the guinea-pig stand out in comparison with the values for the other homogenates examined. 3. The liver, kidney and total brain homogenates of the chicken and the vertebrates display no, or only a very 10w P activity. The highest P activities were measured in the haemolysates. 4. The C and SOD values exhibit a certain parallelism. 5. The peroxide metabolism enzyme activities calculated by utilizing the protein measurements permit the establishment of a more realistic enzymatic activity. INTRODUCTION In an earlier publication (Matkovics et al., 1976) quantitative data measured in organ and tissue homogenates were reported for SOD (EC 1.15.1.1.). This enzyme catalyzes the dismutation of 0 2 anions formed from molecular oxygen in biological systems: O2- + 0 2 + 2 H + - " ) H 2 0 2 + 02
(a)
SOD therefore gives rise to H202, in the decomposition of which the other two enzymes, studied also by us, contained iron-porphyrin skeleton, P (EC 1.11.1.7.) and C (EC 1.11.1.6.), take part. P is able to react with H202 only in the presence of proton donors (b), while the reaction catalyzed by C resembles the over described dismutation reaction
(c): H202 + AH2 ~ 2 H 2 0 + A
(b)
H202 + H202 c__}2 H 2 0 + 02
(C)
Although not only SOD produces H202 in tissues and organs, nevertheless the comprehensive term "peroxide metabolism enzymes" is a fitting one for the enzymes we have studied and described, for they can be intercorrelated in an evolutionary context (Frieden, 1974), and P and C are among the most active enzymes of H202 decomposition; however, it is true that they are not the sole decomposers of H202 in the living world.
The tissues were prepared, and the enzymatic activities were measured and calculated, as described in detail earlier (Matkovics et al., 1976; Part VIII/A). The centrifuged supernatants of the homogenates were used for every enzyme and other measurement. Homogenization was performed in a Potter glass homogenizer, with ice-cooling. SOD was measured quantitatively at pH 10.2, 25°C and 480 nm by the adrenochrome method of Misra & Fridovich (1972). P activities were determined at 25°C and 470 nm by the spectrophotometric guaiacol method of George (1955) (In the book of Methods in Enzymology). The method of Beers & Sizer (1952), involving backmeasurement of the rate of H202 consumption at 240 nm and 25°C, was used for quantitative determination of C activities. Enzymatic activity units were defined previously too (see Part VIII/A). Quantitative protein measurements were made by the method of Lowry et al. (1955), based on the Folin-phenol reagent, bovine plasma albumin being used to prepare the standard curve at 25°C and 675 nm. The reagents employed in enzymatic activity determinations were products of Reanal (Budapest, Hungary), Merck (Darmstadt, GFR) and Calbiochem (Basel, Switzerland) of the highest purity. They could thus be used without preliminary purification. For visible and ultraviolet spectrophotometric measurements, Spektromom 360, Spektromom 2"131(both MOM, Budapest, Hungary) and VSU-2P (Zeiss, Jena, GDR) spectrophotometers were utilized.
MATERIALS AND METHODS
RESULTS AND DISCUSSION
The experimental objects investigated were as follows: fresh-water shell-fish (Anodonta cyfnea), chicken (Gallus domesticus), mouse (Mus musculus, Strain CBA-H2K male), guinea-pig (Carla cutleri), rabbit (Oryctolaxjus cuniculus), cat (Felis catus) and dog (Canis familiaris).
The tissue and organ SOD, P and C values of the experimental animals investigated earlier (the snail, frog, carp, pigeon, rat and pig) were supplemented with peroxide metabolism enzyme measurements on a further 7 animals used mainly also for physiological and pharmacological experimental purposes. These results are given in the tables. It should be noted in general that the tabulated data are the averages of at least 10 measurements. The mean error of the measurements is _4- 5-10~o too.
*Part VIII/B of the series "Properties of enzymes". f To whom reprint requests and correspondence should be addressed (P.O. Box 539, H-6701 Szeged, Hungary). Abbreviations used: as previously and as follows: freshwater shell-fish = f.w.sh.f. (mollusc); guinea-pig = g.p. 397
308
B. MATKOVICS et al. Table 1. Organs kidney liver spleen whole brain lung pancreas thymus f ovarium genital ~ uterus organs ~ testes stomach or chicken gizzard intestine colon • f.w.sh.f. muscle muscles heart muscle skeletal ,muscle )" hemolysate blood l, plasma gills f.w.sh.f, genital organs
f.w.sh.f,
chicken
mouse
715
1060 3370 360 350 300 350
1470 4450 415 565 340 380
120 238 285
g.p. rabbit 1228 5600 635 635 470 630 104 715 635 395
cat
dog
375 3500 500 380 200 300
1200 1760 2950 3370 100 560 300 799 190 560 180 475 395 100 900 96 320 100
176 352 336
300 290
310 272 144
0 650
425
795
500
560
635
300 300 1
280 600 24
256 790 24
305 1500
300 900
313 1600 6
0 715
SOD activities of various animal organ and tissue homogenates, measured at 480 nm and 25°C, and calculated as enzyme units (U) per g wet tissue. In the calculation of the data, a PL/1 p r o g r a m m e was used on a R H O (Robotron, Magdeburg, G D R ) computer. (The problem of computer solution of enzymatic activity calculation will be dealt with in a separate paper in the near future.) Tables 1-3 contain enzymatic activities of only the main tissues and organs, and some endocrine glands.
Because of the very interesting nature of the results obtained to date, problems relating to the peroxide metabolism activities of the endocrine glands in particular will be dealt with in a separate publication. These experimental data suggest that it will be possible to draw certain endocrine evolutionary conclusions.
Table 2. Organs kidney liver spleen whole brain lung pancreas thymus ( ovarium genital ~ uterus organs t, testes stomach or chicken gizzard intestine colon ( f.w.sh.f. | muscle heart muscles } muscle / skeletal I muscle ~ hemolysate blood plasma gills f.w.sh.f, genital organs
f.w.sh.f,
chicken
mouse
2200
0 0 1100 0 2900 30
0 0 2500 0 1900 145 450 500
1000 60
g.p. rabbit 0 0 620 120 1350 60
20 10 800 85 600 75
150 270 300
180 155
cat
dog
180 10 1220 110 1000 120 180
0 0 700 130 1300 90 200 210
400
30 150 90
160 200
20 160 100
750
570
710
90 7500 360 300
82 ---
90
40 70
1600
720
86 20950 280
100 9500 100
90
2400 0
P activities of various animal organ and tissue homogenates (U)g wet tissue.
450
Animal peroxide metabolism enzymes
399
Table 3. Organs kidney liver spleen whole brain lung pancreas thymus genital )" ovarium uterus organs ~ testes stomach or chicken gizzard intestine colon r f.w.sh.f. | muscle muscles ~ heart | muscle | skeletal 1. muscle hemolysate blood plasma gills f.w.sh.f, genital organs
f.w.sh.f,
chicken
0.220
0.265 2.010 0.260 0.020 0.119 0.320
mouse
g.p. rabbit cat
dog
9.600 4.800 2.400 0.320 8.055 10.500 2.140 2.200 3.340 1.458 1.175 1.580 1.600 0.660 1.500 0.070 1.104 0.040 0.060 0.100 0.195 1.860 0.480 0.598 2.000 0.210 0.960 0.200 0.120 1.965 0.800 0.582 0.940 0.240 - 1.000 0.840 0.120 0.940 0.205 0.104 0.123
0.142 0.060
---
0.153
0.195
1.660 - 0.700 - 0.420
-0.695 0.400 0.500 0.410 0.240
0.009
0.100 4.020 0.0
0.580 0.230 0.324 0.600
0.090 0.244 0.070 0.152 0.256 90.000 11.400 0.600 - - 11.555 0.040 0.182 0.030 - 0.195
0.082 0.100
C activities of various animal organ and tissue homogenates (B.U./g wet tissue). Table 1 lists the SOD values. For the mollusc, only the branchia, kidney, plantaris and stomach data are given. In place of the stomach data, those of the gizzard are listed for the chicken. (Our data relating to human tissues up to now from normal biopsies are only few to be given in a separate column.) Analysis of the data in Table 1 leads to the following conclusions regarding the SOD values of the organs and tissues examined: (a) As in the earlier results (Part VIII/& 1976) here too the liver exhibits the highest SOD activities. The guinea-pig liver has an outstandingly high SOD activity, while the values for the dog, chicken and rabbit livers are very similar to one another. (b) In most cases the kidney homogenates have SOD activities about 3 times lower than those of the liver; in some cases (chicken and dog), these activities approach those of the hemolysates, which are all of nearly the same magnitude. The blood plasma exhibits no, or only a very low activity of SOD (this may perhaps be correlated with the slight spontaneous hemolysis of the red blood cells). (c) The SOD activities of the other tissue and organ homogenates lie in the range 50-800 U/g wet tissue. Similarly, conclusions may be drawn from the quantitative P results in Table 2: (a) The highest P activities in the red blood cell hemolysates were found in the chicken, mouse and rabbit, the chicken exhibiting the largest values. No P activity was observed in the red blood cells of the dog and guinea-pig. The plasma P activities lie in the range 100450 U/g. (b) The highest P activities are found in the organs of the chicken, while the P occurs to the greatest extent in the tissue and organ homogenates of the g.p., cat and dog.
(c) There is in general no, or only low P activity in the liver and kidney tissues. The kidney and branchia of the mollusc exhibit high P activities. (d) The activity sequence for the various tissue and organ homogenates is as follows: hemolysate > lung > spleen > myocardium. Table 3 gives the C activities in B.U./g wet tissue. Here too, several points may be noted: (a) The highest C activities are those of the hemolysates, that of the mouse blood hemolysate being outstanding at 90.0 B.U./g wet tissue. (b) The most complete values are those relating to the organ and tissue homogenates of the guinea-pig. (c) The C activity sequence is: hemolysate > liver > kidney > spleen > lung. (d) The C activities of the other organ and tissue homogenates lie in the range 0.009-1.00 B.U./g wet tissue, the only exception being that of the homogenate of the g.p. ovary (1.660 B.U./g wet tissue). (e) The C values for all of the brain tissue homogenates studied are markedly low. (We shall deal with this problem separately in the description of the specific activities.) If the C activities of the organ and tissue homogenates are made the subject of a general study, then it can be seen at once that the C activities provide a picture of the aerobic metabolism of the tissues. More exactly, the C activity values are higher in those tissues more strongly connected with the aerobic metabolism and with molecular oxygen, or carrying out active protein synthesis. In Table 4 we wish to put our studies to date in a new light. This table contains our measurements on tissue proteins reported by Lowry et al. (1951);
400
B. MATKOV1CSet al. Table 4. Organs kidney liver spleen whole brain lung pancreas genital ~" ovarium uterus organs ( testes stomach or chicken gizzard intestine colon • f.w.sh.f. muscle heart muscles muscle skeletal muscle blood hemolysate • plasma gills f.w.sh.f, genital organs
f.w.sh.f,
chicken
mouse
g.p.
dog
50
50 150 70 36 70 78
142.8 196 93.2 48 88 91
56 140 67 42 63 77 46 32 45
70 90 50 43 65 72 50 40
36 41 37
40 43 35
45
46
57.2 47 45 20 45
124
45 120* 150"
58.4 130* 110"
40
45
110"
100"
130"
128'
16 38
Protein contents of various animal organ and tissue homogenates (mg protein/g wet tissue)• these are later used to present the specific activities of the peroxide metabolism enzymes studied. In our protein determinations the results are given in mg protein/g wet tissue. Protein values were determined for organ and tissue homogenates from mollusc, mouse, chicken, g.p. and dog. In the following tables we shall see the specific activities of the peroxide metabolism enzymes for the animals listed in Table 4, in enzyme units/mg protein. Table 5 gives the specific enzymatic activities for the mollusc. In this and the subsequent tables the specific activity sequence will be SOD, P and C. In the mollusc organ examinations, no special effort was made to prepare the ganglia, for example, but the specific activities of the peroxide metabolism enzymes were investigated on only four easily preparable organs. The tables are interesting in that there are modifications in the sequences of enzymatic activities relating to the organ and tissue homogenates as established earlier in connection with the specific enzymatic activities; this is due to the increase in the Table 5. Organs kidney genital organs muscles gills
SOD
P
C
14.3
44
0.0044
18.8 0 0.0030 0 2.0 0.0004 0 150 0.0050
Specific peroxide metabolism enzymatic activities of mollusc organ and tissue homogenates (enzyme U/mg protein).
value of the denominator in the quotient. For instance, in Table 5 the specific S O D activities of the reproductive system precede the values measured for the kidney homogenates. The specific P and C activities are highest of all for the mollusc branchia. Table 6 lists the specific enzymatic activities relating to the mouse tissues. It is interesting to observe that the brain lies in third place, after the liver and kidney, in the sequence of S O D specific activities, standing out from the moderate S O D activities exhibited by the other organ and tissue homogenates, which barely differ from one another. The mouse brain, liver and kidney do not exhibit P activity. The high specific P activity of the hemolysate is followed directly by that of the spleen. Table 6. Organs
SOD
P
C
kidney liver spleen whole brain lung pancreas testes
12.09 22.47 4.64 12.05 3.85 3.19 1.085
0 0 31.0 0 21.1 1.67 9.24
0.072 0.055 0.0129 0.015 0.002 0.0023 0.0033
4.25
17.7
0.002
4.83 4.6 0.2
1.58 73.2 0.9
0.0012 0.6923 0.0003
muscles blood
[" heart ,) muscle ] skeletal t muscle hemolysate plasma
Specific peroxide metabolism enzymatic activities of mouse organ and tissue homogenates (U/mg protein).
Animal peroxide metabolism enzymes
401 Table 9.
Table 7. Organs
SOD
P
C
kidney liver spleen whole brain lung pancreas intestine
21.2 22.5 5.1 9.7 4.3 4.5 6.3
0.0 0.0 15.7 0.0 41.4 0.4 1.3
0.005 0.013 0.004 0.001 0.002 0.004 0.001
14.4
1.6
0.003
1.9 139.7 2.3 21.3
0.002 0.026 0.0 0.003
muscles blood
heart muscle skeletal muscle hemolysate plasma
gizzard
6.6 2.0 0.008 5.1
Specific peroxide metabolism enzymatic activities of chicken organ and tissue homogenates (U/mg protein). In comparison with the outstanding specific C activity of the hemolysate, the mouse liver and kidney have moderate activities, and the other homogenates low values. Table 7 presents the specific peroxide metabolism enzyme values for the organ and tissue homogenates of the chicken. As regards the SOD specific enzymatic activities, those of the liver, kidney and myocardium (in decreasing order) exceed 10 U/mg protein. Of the P specific activities, here too the outstanding value for the hemolysate is followed by high values for the lung, gizzard and spleen. C exhibits high activities in the hemolysate and liver, and low activities in the other homogenates examined. In Table 8 we find the specific activities of the peroxide metabolism enzymes in the organs and tissues of the guinea-pig. In order of magnitude, SOD activities of more than 10U/mg protein are exhibited by the liver, the adrenal, the kidney, the myocardium, the uterus, the ovary and the brain. The SOD activity of the liver homogenate here is 40.0 U/mg protein. Table 8. Organs
SOD
P
C
kidney liver spleen whole brain lung pancreas thymus genital )- ovarium uterus organs ~ testes stomach intestine colon hemolysate blood plasma
21.9 40.0 9.5 15.1 7.5 8.2 4.5 15.5 19.8 8.8 4.9 8.6 9.1 6.1 0.2
0.0 0.0 9.3 2.9 21.4 0.8 19.6 3.2 8.4 6.7 0.8 3.7 2.4 0.0 3.3
0.0857 0.0153 0.0236 0.0025 0.0295 0.0124 0.0347 0.0204 0.0263 0.0023 0.0461 0.0171 0.0114 0.0877 0.0017
Specific peroxide metabolism enzymatic activities of guinea-pig organ and tissue homogenates (U/mg protein). C.B.P. 56/4B--D
Organs
SOD
P
C
kidney liver spleen whole brain lung pancreas genital J" ovarium organs ~ uterus stomach intestine colon heart muscle muscles skeletal muscle hemolysate blood plasma
25.1 37.4 11.2 18.6 8.6 6.6 18.0 8.0 7.8 6.3 4.1
0.0 0.0 14.0 3.0 20.0 1.3 4.0 5.3 0.5 3.7 2.9
0.1151 0.0162 0.0300 0.0023 0.0307 0.0273 0.0200 0.0235 0.0173 0.0116 0.0069
13.8
15.4
0.0130
7.0 12.5 0.06
3.0 0.0 4.5
0.0057 0.0903 0.0020
Specific peroxide metabolism enzymatic activities of dog organ and tissue homogenates (U/rag protein). The sequence for the higher P specific activities is headed by the lung and the thymus, with the myocardium and spleen in third and fourth places. The kidney, adrenal, liver and hemolysate do not exhibit P activity. The C values in Table 8 lead to the following findings: (a) The C activity of the adrenal generally exceeds the highest C activity of the hemolysate, which is also approached by that of the kidney homogenate. (b) The other measured C activities lie in the range 0.0017-O.0461 B.U./mg protein. Table 9 lists the specific activities of the peroxide metabolism enzymes in the organs and tissues of the dog. As regards the SOD activities, the highest is that of the liver here too, followed by that of the adrenal. The P specific activity sequence is the lung, the myocardium and the spleen. The highest specific C activity is found in the adrenal. To give a brief summary of all the above data, it may be said, mainly from a comparison of the results relating to the enzyme U/g wet tissue and the specific enzyme U/mg protein values, that the enzymatic activities obtained via quantitative protein determinations provide a new picture in the comparison of the peroxide metabolism enzymes. Both sets of data have their own value. The more extreme values of the enzymatic activities calculated per g of wet tissue are rather conspicuous, whereas the specific activities are more realistic.
REFERENCES
BEERSR. F. JR. • SIZER I. W. (1952) Spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase, d. biol. Chem. 195, 133-140. COLOWICKS. P. & KAPLAr~N. O. (1955) Methods in Enzymology, Vol. 2. pp. 764-775. Academic Press, New York. FRmOEN E. (1974) Biochemical evolution of iron and copper proteins. Chem. & Eng. News. March 25, pp. 42-46.
402
B. MATKOV1CSet al.
LOWRY O. H., ROSEBROUGHN. J., FARR A. L. RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MATKOVICS B., NOV/~K R., HOANG DUC HANH, SZAB6 L., VARGA Sz. I. • ZALESNAG. (1976) A comparative study of some more important experimental animal peroxide
metabolism enzymes. (VIII/A) Comp. Biochem. Physiol. (In press.) MISRA H. P. & FRIDOVICH I. (1972) The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J. biol. Chem. 247, 3170-3175.
