BIOCHEMICAL
MEDICINE
AND
METABOLIC
BIOLOGY
43, 159-162 (19%))
Occurrence and Comparison of Sulfite Oxidase Activity in Mammalian Tissues FRANCESC CABR~?, CARME MAR~N, MARTA AND ENRIC I. CANELA Department
of Biochemistry
and Physiology, Barcelona,
University Catalonia,
of Barcelona, Spain
CASCANTE,
Marti
i Franqu?s
1, E-08071
Received August 8, 1989, and in revised form January 5, 1990
Sulfite oxidase (EC 1.8.3.1) acts as the last enzyme in the oxidative degradation pathway of sulfur amino acids (1). This enzyme also plays a role in preventing the toxic effects of sulfur dioxide (2). Sulfite oxidase is located in the intermembranous space of mitochondria (3) and it is found in most tissues of the body (435). Patients with severe cases of sulfite oxidase deficiency suffer from mental retardation, eye problems, and early death. Less severe cases can result in intolerance to some sulfite-containing foods. Other aspects of the deficiency of sulfite oxidase are related to the metabolism of sulfur dioxide and sulfite. Accumulation of endogenously generated sulfite and S-sulfonate compounds in the respiratory tract and in the plasma of rats is inversely proportional to their hepatic sulfite oxidase activity (6-8). Toxicity caused by endogenous sulfite can be related to metabolism of sulfur-containing amino acids (see Ref. (9) for review). In the present study the occurrence of sulfite oxidase activity in several tissues of various mammalian species is reported and the results are discussed according to a possible function of this enzyme. MATERIALS
AND METHODS
Materials Cytochrome c (type III) was obtained from Sigma and sodium sulfite from Merck (Darmstad, FRG). All other chemicals and biochemicals obtained from commercial sources were of analytical grade. Stock solutions of 0.01 M sodium sulfite were prepared in ultrapure water (MilliQ-Millipore Waters) and stored frozen until use. Animals The animals used in this work were provided by a local slaughterhouse Barcelona and the laboratory animals service of the University of Barcelona. all cases tissues were obtained within 24 hr after sacrifice.
in In
159 0885-4505/90 $3.00 Copyright Q 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
160
CABRB ET AL.
Determination
of Activities
Sulfite oxidase activity was measured at 30°C by monitoring the reduction of cytochrome c at 550 nm in a PYE Unicam SP 1800 speai’ophotometer according to the method of Kessler and Rajagopalan (10). We defined 1 unit of sulfite oxidase as the amount of enzyme required to reduce 1 pmole of substrate/min (under the assay conditions). The molar absorptivity of cytochrome c was taken to be 2.11 x lo4 M-’ cm-’ (6,7). Preparation
of Enzyme Extracts
Acetone powder from 150 g of tissue was prepared according to the method of Cohen and Fridovich (11). Acetone powder was resuspended in 0.05 M sodium phosphate buffer, pH 7.8, containing 1 x 10m4M EDTA, in a ratio of 10 ml of buffer/l g of powder and stirred for 30 min at 4°C. The suspension was centrifuged for 10 min (18,OOOg,4°C). Then, 2.5 ml of the supernatant was desalted by passage through a Sephadex G-25 (Pharmacia) column (1 x 5 cm), equilibrated with Tris(50 mM)-HCI, pH 7.8. The active fractions eluted were collected and applied to a column (1.6 x 1.5 cm) of DEAE-Sepharose Fast Flow (Pharmacia) equilibrated with the same buffer. After several washings with the same buffer, the enzyme was eluted with 1 M NaCl in Tris(O.OSM)-HCl, pH 7.8. Active fractions were pooled and total sulfite oxidase activity present was then quantified, as described above. Statistical
Analysis of Data
Multifactor ANOVA and F test were performed with Statgrafics. RESULTS
AND DISCUSSION
Brain, spleen, lung, and testis exhibited very low activities of sulfite oxidase, which were hardly measurable under the assay conditions (Table 1). In contrast liver, kidney, and heart tissues exhibited high activities of sulfite oxidase. These levels of sulfite oxidase activity could be related to the degree of amino acid TABLE 1 Sulfite Oxidase Activity, Expressed in Units per Gram of Tissue Extracted” w Liver Kidney Heart Brain Spleen Lung Testis
0.07 1.33 0.32 0.47
+ f + 2 ** ** 0.048 k
Sheep
Bovine
Horse
0.01 0.63 2 0.07 0.71 2 0.08 0.73 + 0.05 0.11 0.49 f 0.05 0.07 + 0.08 0.57 2 0.03 0.03 0.11 + 0.01 0.47 + 0.07 N.D. 0.02 N.D. N.D. N.D. 0.08 + 0.009 0.007 rt 0.001 N.D. 0.013 f 0.002 (1.6 f 0.2)x 1O-4 N.D. 0.004 N.D. 0.21 f 0.03 **
Rat
Rabbit
1.52 f 0.1 0.25 + 0.03 N.D. 0.29 k 0.01 1.26 + 0.2 ** 0.02 _’ 0.001 ** 0.04 k 0.001 ** 0.039 + 0.006 ** 0.096 f 0.008 **
’ One unit is the amount of sulfite oxidase required to reduce 1 Imole of cytochrome c per minute (see Materials and Methods). ND, sulfite oxidase activity is not detected. **, Sulfite oxidase activity was not tested in this tissue, since it was not obtained tissue extracts in good conditions. Values are given as means f r(O.05) x SE.
SULFITE
OXIDASE
IN MAMMALIAN
TISSUES
161
catabolism in each tissue. High enzyme activity in the liver may be due to the oxidation of sulfite generated by normal sulfur amino acid catabolism in this organ. Kidney uses glucose, fatty acids, ketonic compounds, and also amino acids to meet its energy requirements. So, sulfite oxidase in kidney could play a role in oxidizing eithr sulfite generated by amino acid catabolism in this organ or sulfite present in blood provided from the degradation of amino acids in other organs which lack this enzyme. The high level of sulfite oxidase in heart could be related to the rapid metabolism of amino acids in this muscle due to its high turnover of proteins. The low value obtained for sulfite oxidase in lung does not exclude the possibility that the enzyme plays a role in preventing the toxic effects of sulfur dioxide respired propounded by Cohen et al. (2), since the intake of sulfite as a result of inhaling SO2 is usually lower than the intake by oral ingestion. It is interesting to note that data on the levels of sulfite oxidase activity in mammalian tissues are diverse. The highest level of enzymatic activity detected in liver agrees with sulfite oxidase activity in normal human liver reported by several authors (12,13), and the lowest level found in lung agrees with low activity of enzyme found in human lung by BeckSpeier et al. (13). Multivariate analysis of variance followed by F test of the results of Table 1 shows that the species dependence of sulfite oxidase is minor (38%) when compared to the tissue dependence (94%). The number of different mammalian species tested is enough to consider valid the tendency of some organs to have high levels of sulfite oxidase, and that of other tissues to have low levels of the enzyme (Table 1). In conclusion, our data suggest that significant differences in levels of sulfite oxidase in mammalian tissue exist. The presence of sulfite oxidase activity in most mammalian tissues ratifies the fact that detoxification of sulfite in these tissues is a prominent process. We provide evidence for the significance of sulfite oxidase in a number of tissues and that the enzyme may play an important role in nonpathologic conditions. SUMMARY Tissue extracts from six mammalian species have been assayed for sulfite oxidase (sulfite: ferricytochrome c oxidoreductase, EC 1A.3.1) activity with cytochrome c as electron acceptor. Our results show a large distribution of sulfite oxidase activity in mammalian tissues. Liver, kidney, and heart tissues exhibit high activities whereas brain, spleen, and testis show very low activities. No significant species dependence was observed for the activity of this enzyme. REFERENCES 1. Johnson, J. L., and Rajagopalan, K. V., J. C/in. Invest. 58, 551 (1976). 2. Cohen, H. J., Drew, R. T., Johnson, J. L., and Rajagopalan, K. V., Proc. Nat/. Acad. Sci. USA 70, 3655 (1973). 3. Rajagopalan, K. V., in “Molybdenum and Molybdenum-Containing Enzymes” (M. P. Coughlan, Ed.), p. 242. Pergamon Press, New York, 1980. 4. MacLeod, R. M., Farkas, W., Fridovich, I., and Handler, P., J. Bid. Chem. 236, 1841 (1961). 5. Johnson, J. L., Jones, P. H., and Rajagopalan, K. V., J. Biol. Chem. 252, 4994 (1977). 6. Gunnison, A. F., and Jacobsen, D. W. CRC Cd. Rev. Toxicol. 17, 185 (1987).
162
CABRfi ET AL.
7. Gunnison, A. F., Sellakumar, A., Currie, D., and Snyder, E. A., .I. Toxicol. Environ. Health 21, 141 (1987). 8. Gunnison, A. F., Sellakumar, A., Snyder, E. A., and Currie, D., Environ. Res. 46, 59 (1988). 9. Cooper, A. J. L., Annu. Rev. Biochem. 52, 187 (1983). 10. Kessler, D. L., and Rajagopalan, K. V., J. Biol. Chem. 247, 6566 (1972). 11. Cohen, H. J., and Fridovich, I., J. Biol. Chem. 246, 359 (1971). 12. Johnson, J. L., Waud, W. R., Rajagopalan, K. V., Duran, M., Beemer, F. A., and Wadman, S. K., Proc. Natl. Acad. Sci. USA 77, 3715 (1980). 13. Beck-Speier, I., Hinze, H., and Holzer, H., Biochim. Biophys. Acta 841, 81 (1985).
MEDICINE
AND
METABOLIC
BIOLOGY
43, 159-162 (19%))
Occurrence and Comparison of Sulfite Oxidase Activity in Mammalian Tissues FRANCESC CABR~?, CARME MAR~N, MARTA AND ENRIC I. CANELA Department
of Biochemistry
and Physiology, Barcelona,
University Catalonia,
of Barcelona, Spain
CASCANTE,
Marti
i Franqu?s
1, E-08071
Received August 8, 1989, and in revised form January 5, 1990
Sulfite oxidase (EC 1.8.3.1) acts as the last enzyme in the oxidative degradation pathway of sulfur amino acids (1). This enzyme also plays a role in preventing the toxic effects of sulfur dioxide (2). Sulfite oxidase is located in the intermembranous space of mitochondria (3) and it is found in most tissues of the body (435). Patients with severe cases of sulfite oxidase deficiency suffer from mental retardation, eye problems, and early death. Less severe cases can result in intolerance to some sulfite-containing foods. Other aspects of the deficiency of sulfite oxidase are related to the metabolism of sulfur dioxide and sulfite. Accumulation of endogenously generated sulfite and S-sulfonate compounds in the respiratory tract and in the plasma of rats is inversely proportional to their hepatic sulfite oxidase activity (6-8). Toxicity caused by endogenous sulfite can be related to metabolism of sulfur-containing amino acids (see Ref. (9) for review). In the present study the occurrence of sulfite oxidase activity in several tissues of various mammalian species is reported and the results are discussed according to a possible function of this enzyme. MATERIALS
AND METHODS
Materials Cytochrome c (type III) was obtained from Sigma and sodium sulfite from Merck (Darmstad, FRG). All other chemicals and biochemicals obtained from commercial sources were of analytical grade. Stock solutions of 0.01 M sodium sulfite were prepared in ultrapure water (MilliQ-Millipore Waters) and stored frozen until use. Animals The animals used in this work were provided by a local slaughterhouse Barcelona and the laboratory animals service of the University of Barcelona. all cases tissues were obtained within 24 hr after sacrifice.
in In
159 0885-4505/90 $3.00 Copyright Q 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
160
CABRB ET AL.
Determination
of Activities
Sulfite oxidase activity was measured at 30°C by monitoring the reduction of cytochrome c at 550 nm in a PYE Unicam SP 1800 speai’ophotometer according to the method of Kessler and Rajagopalan (10). We defined 1 unit of sulfite oxidase as the amount of enzyme required to reduce 1 pmole of substrate/min (under the assay conditions). The molar absorptivity of cytochrome c was taken to be 2.11 x lo4 M-’ cm-’ (6,7). Preparation
of Enzyme Extracts
Acetone powder from 150 g of tissue was prepared according to the method of Cohen and Fridovich (11). Acetone powder was resuspended in 0.05 M sodium phosphate buffer, pH 7.8, containing 1 x 10m4M EDTA, in a ratio of 10 ml of buffer/l g of powder and stirred for 30 min at 4°C. The suspension was centrifuged for 10 min (18,OOOg,4°C). Then, 2.5 ml of the supernatant was desalted by passage through a Sephadex G-25 (Pharmacia) column (1 x 5 cm), equilibrated with Tris(50 mM)-HCI, pH 7.8. The active fractions eluted were collected and applied to a column (1.6 x 1.5 cm) of DEAE-Sepharose Fast Flow (Pharmacia) equilibrated with the same buffer. After several washings with the same buffer, the enzyme was eluted with 1 M NaCl in Tris(O.OSM)-HCl, pH 7.8. Active fractions were pooled and total sulfite oxidase activity present was then quantified, as described above. Statistical
Analysis of Data
Multifactor ANOVA and F test were performed with Statgrafics. RESULTS
AND DISCUSSION
Brain, spleen, lung, and testis exhibited very low activities of sulfite oxidase, which were hardly measurable under the assay conditions (Table 1). In contrast liver, kidney, and heart tissues exhibited high activities of sulfite oxidase. These levels of sulfite oxidase activity could be related to the degree of amino acid TABLE 1 Sulfite Oxidase Activity, Expressed in Units per Gram of Tissue Extracted” w Liver Kidney Heart Brain Spleen Lung Testis
0.07 1.33 0.32 0.47
+ f + 2 ** ** 0.048 k
Sheep
Bovine
Horse
0.01 0.63 2 0.07 0.71 2 0.08 0.73 + 0.05 0.11 0.49 f 0.05 0.07 + 0.08 0.57 2 0.03 0.03 0.11 + 0.01 0.47 + 0.07 N.D. 0.02 N.D. N.D. N.D. 0.08 + 0.009 0.007 rt 0.001 N.D. 0.013 f 0.002 (1.6 f 0.2)x 1O-4 N.D. 0.004 N.D. 0.21 f 0.03 **
Rat
Rabbit
1.52 f 0.1 0.25 + 0.03 N.D. 0.29 k 0.01 1.26 + 0.2 ** 0.02 _’ 0.001 ** 0.04 k 0.001 ** 0.039 + 0.006 ** 0.096 f 0.008 **
’ One unit is the amount of sulfite oxidase required to reduce 1 Imole of cytochrome c per minute (see Materials and Methods). ND, sulfite oxidase activity is not detected. **, Sulfite oxidase activity was not tested in this tissue, since it was not obtained tissue extracts in good conditions. Values are given as means f r(O.05) x SE.
SULFITE
OXIDASE
IN MAMMALIAN
TISSUES
161
catabolism in each tissue. High enzyme activity in the liver may be due to the oxidation of sulfite generated by normal sulfur amino acid catabolism in this organ. Kidney uses glucose, fatty acids, ketonic compounds, and also amino acids to meet its energy requirements. So, sulfite oxidase in kidney could play a role in oxidizing eithr sulfite generated by amino acid catabolism in this organ or sulfite present in blood provided from the degradation of amino acids in other organs which lack this enzyme. The high level of sulfite oxidase in heart could be related to the rapid metabolism of amino acids in this muscle due to its high turnover of proteins. The low value obtained for sulfite oxidase in lung does not exclude the possibility that the enzyme plays a role in preventing the toxic effects of sulfur dioxide respired propounded by Cohen et al. (2), since the intake of sulfite as a result of inhaling SO2 is usually lower than the intake by oral ingestion. It is interesting to note that data on the levels of sulfite oxidase activity in mammalian tissues are diverse. The highest level of enzymatic activity detected in liver agrees with sulfite oxidase activity in normal human liver reported by several authors (12,13), and the lowest level found in lung agrees with low activity of enzyme found in human lung by BeckSpeier et al. (13). Multivariate analysis of variance followed by F test of the results of Table 1 shows that the species dependence of sulfite oxidase is minor (38%) when compared to the tissue dependence (94%). The number of different mammalian species tested is enough to consider valid the tendency of some organs to have high levels of sulfite oxidase, and that of other tissues to have low levels of the enzyme (Table 1). In conclusion, our data suggest that significant differences in levels of sulfite oxidase in mammalian tissue exist. The presence of sulfite oxidase activity in most mammalian tissues ratifies the fact that detoxification of sulfite in these tissues is a prominent process. We provide evidence for the significance of sulfite oxidase in a number of tissues and that the enzyme may play an important role in nonpathologic conditions. SUMMARY Tissue extracts from six mammalian species have been assayed for sulfite oxidase (sulfite: ferricytochrome c oxidoreductase, EC 1A.3.1) activity with cytochrome c as electron acceptor. Our results show a large distribution of sulfite oxidase activity in mammalian tissues. Liver, kidney, and heart tissues exhibit high activities whereas brain, spleen, and testis show very low activities. No significant species dependence was observed for the activity of this enzyme. REFERENCES 1. Johnson, J. L., and Rajagopalan, K. V., J. C/in. Invest. 58, 551 (1976). 2. Cohen, H. J., Drew, R. T., Johnson, J. L., and Rajagopalan, K. V., Proc. Nat/. Acad. Sci. USA 70, 3655 (1973). 3. Rajagopalan, K. V., in “Molybdenum and Molybdenum-Containing Enzymes” (M. P. Coughlan, Ed.), p. 242. Pergamon Press, New York, 1980. 4. MacLeod, R. M., Farkas, W., Fridovich, I., and Handler, P., J. Bid. Chem. 236, 1841 (1961). 5. Johnson, J. L., Jones, P. H., and Rajagopalan, K. V., J. Biol. Chem. 252, 4994 (1977). 6. Gunnison, A. F., and Jacobsen, D. W. CRC Cd. Rev. Toxicol. 17, 185 (1987).
162
CABRfi ET AL.
7. Gunnison, A. F., Sellakumar, A., Currie, D., and Snyder, E. A., .I. Toxicol. Environ. Health 21, 141 (1987). 8. Gunnison, A. F., Sellakumar, A., Snyder, E. A., and Currie, D., Environ. Res. 46, 59 (1988). 9. Cooper, A. J. L., Annu. Rev. Biochem. 52, 187 (1983). 10. Kessler, D. L., and Rajagopalan, K. V., J. Biol. Chem. 247, 6566 (1972). 11. Cohen, H. J., and Fridovich, I., J. Biol. Chem. 246, 359 (1971). 12. Johnson, J. L., Waud, W. R., Rajagopalan, K. V., Duran, M., Beemer, F. A., and Wadman, S. K., Proc. Natl. Acad. Sci. USA 77, 3715 (1980). 13. Beck-Speier, I., Hinze, H., and Holzer, H., Biochim. Biophys. Acta 841, 81 (1985).
