/ . Biochem. 86, 871-881 (1979)
and Ketones from Guinea Pig Liver Hideo SAW ADA, Akira HARA, Fumiko KATO, and Toshihiro NAKAYAMA Gifu College of Pharmacy, Mitahora-higashi, Gifu, Gifu 502 Received for publication, February 10, 1979
NADPH-dependent enzymatic reduction of aromatic aldehydes and ketones observed in the cytosol of guinea pig liver was mediated by at least three distinct reductases (AR 1, AR 2, and AR 3), which were separated by DEAE-cellulose chromatography. By several procedures AR 2 and AR 3 were purified to homogeneity, but AR 1 could be purified only 30-fold because of the small amount. These enzymes were found to have similar molecular weights of 34,000 to 36,000 and similar Stokes radii of about 2.5 nm. AR 3 was identical to aldehyde reductase [EC 1.1.1.2] in substrate specificity for aromatic aldehydes and Dglucuronate and specific inhibition by barbiturates. AR 1 and AR 2 acted on aromatic ketones and cyclohexanone as well as aromatic aldehydes at optimal pHs of 5.4 and 6.0, respectively, and were immunochemically distinguished from AR 3. AR 1 was the most sensitive to sulfhydryl reagents, and AR 2 was more stable at 50°C than the other enzymes. Similar heterogeneity was observed in the kidney enzymes, but other tissues had little aldehyde reductase activity and contained only AR 3. In addition, lung contained a high molecular weight aromatic ketone reductase different from the above reductases.
Biological reduction of biogenic and xenobiotic carbonyl compounds to their corresponding alcohols is mediated by several cytoplasmic pyridine nucleotide-dependent oxidoreductases. Alcohol dehydrogenase catalyzes the reduction of aldehydes, alicyclic ketones, and some steroids in the presence of NADH (1-3). NADPH-dependent aldehyde reductase catalyzes the reduction of aldehydes and some aldoses, but is generally inactive towards ketones (4-10). However, aldehyde reductases differing in substrate specificity have been isolated from several mammalian species. The pig kidney Abbreviations: AR 1, aldehyde reductase 1; AR 2, aldehyde reductase 2; AR 3, aldehyde reductase 3. Vol. 86, No. 4, 1979
enzyme is active on a-diketones (4), whereas the enzymes from other animal tissues reduce only aldehydes (5-9). Four multiple molecular forms of this enzyme have been isolated from human brain and two of them have the ability to reduce several aromatic ketones (10). In addition, a high molecular weight aldehyde reductase with high reactivity for cyclohexanone and D-ribose has been observed in several mammalian livers (11). On the other hand, the enzymatic reduction of a number of ketones has been reviewed (12), but the reductases responsible for this have rarely been purified except for the reductase for aromatic aldehydes and ketones from rabbit kidney (13) and a,/9-unsaturated ketone reductase from human liver and dog
871
Downloaded from https://academic.oup.com/jb/article-abstract/86/4/871/770838 by Mount Sinai Icahn School of Medicine user on 03 February 2019
Purification and Properties of Reductases for Aromatic Aldehydes
H. SAWADA, A. HARA, F. KATO, and T. NAKAYAMA
872
EXPERIMENTAL PROCEDURE Materials—All chemicals were of reagent grade and were purchased from commercial sources. p-Nitrobenzyl alcohol and p-nitroacetophenone were recrystallized from ethanol. Phenyl-(jpyridyl)carbinol was synthesized from 4-benzoylpyridine by addition of an excess of NaBH4 in methanol and recrystallized from chloroform. Proteins used for Sephadex column and polyacrylamide gel calibration were purchased from Sigma Chemical Co. and Seikagaku Kogyo Co., respectively. Human prostatic acid phosphatase was isolated by the method reported previously (17). Sephadex G-75 and G-100 were obtained from Pharmacia Fine Chemicals and DEAE-cellulose (DE32) from Whatman. Cibacron-substituted Sephadex and hydroxylapatite were prepared by the methods of Bohme et al. (18) and Levine (19), respectively. Buffers—The following buffers were used. Buffer A, 10 mM Tris-HCl buffer containing 5 mM 2-mercaptoethanol and 0.5 mM EDTA, pH7.5; Buffer B, 10 mM Tris-HCl buffer containing 5 mM 2-mercaptoethanol and 1 mM K2HPO4, pH7.5; Buffer C, 50 mM Tris-HCl buffer containing 1 mM 2-mercaptoethanol and 0.15 M KC1, pH 7.5; Buffer D, 50 mM Tris-HCl buffer (pH 8.5), 5 mM 2mercaptoethanol, 0.5 mM EDTA, and 0.6 M ammonium sulfate (adjusted to pH 8.5 with 28 % NH4OH). All pH titrations were performed at 25°C. Enzyme Assay—Enzyme activity was measured by the rate of NADPH oxidation monitored at 340 nm. Unless otherwise specified, the assay
mixture contained, in a total volume of 2.5 ml, 80 mM phosphate buffer (pH 6.0) or citrate buffer (pH 5.4), 0.08 mM NADPH, an appropriate amount of enzyme preparation, and 2.5 / glucuronate and p-nitroacetophenone as substrates. In gel filtration both activities of all the tissues except for lung gave a single broad peak at a similar low molecular weight of about 34,000, but it was found that, in addition to the low molecular weight active peak, lung also contained a high molecular weight active peak that had only a high p-nitroacetophenone reductase activity. Figure 7 shows elution profiles of the reductase activities of the low and high molecular weight fractions from each tissue on DE32 chromatography. Activities of aldehyde reductases in kidney showed a similar elution pattern to that in liver (Fig. 1), but the low molecular weight reductases of lung, brain, and heart (data not shown) gave a single active peak that corresponded to liver AR 3. The high molecular weight reductase of lung was eluted at a
Vol. 86, No. 4, 1979
Downloaded from https://academic.oup.com/jb/article-abstract/86/4/871/770838 by Mount Sinai Icahn School of Medicine user on 03 February 2019
Fig. 8. Immunological characterization of aldehyde reductases by the Ouchterlony double-diffusion technique. Plate A, center well contained anti-AR 3 serum, peripheral wells contained the following; (1), AR 3; (2), AR 2; (3), AR 1; (4), enzyme preparation from the Sephadex G-100 step. Plate B, center well contained anti-AR 3 serum, peripheral wells contained AR 3-fractions of DE32 chromatography from lung (1), kidney (2), heart (3), and brain (4).
different NaCl concentration from the other reductases. Double Diffusion Gel Precipitations—Antisera against AR 3 from guinea pig liver were produced in rabbits. By use of the Ouchterlony double diffusion test (Fig. 8A), the antisera gave a single and sharp precipitin line against the antigens and the liver enzyme preparation from the Sephadex G-100 step, but no precipitin line was observed with AR 1 and AR 2. Immunodiffusion against the AR 3-fractions obtained from DE32 chromatography of enzymes from different tissues of the same species resulted in a single precipitin line with the antisera against liver AR 3, and the lines fused completely with each other (Fig. 8B). DISCUSSION We have demonstrated the existence in guinea pig liver of at least three distinct NADPH-dependent enzymes which catalyze the reduction of aldehydes to alcohols, and have presented procedures by which the three reductases (AR 1, AR 2, and AR 3) can be isolated from the cytosol of guinea pig liver. Our AR 2 and AR 3 preparations were judged to be pure by polyacrylamide disc gel electrophoresis with and without sodium dodecyl sulfate and an agarose diffusion test with rabbit anti-AR 3 serum, but AR 1 contained a minor
880
H. SAWADA, A. HARA, F. KATO, and T. NAKAYAMA
In spite of many reports on enzyme activities reducing aromatic aldehydes and ketones, as reviewed by Bachur (12), the reductases responsible for ketones have not been purified sufficiently yet. Culp and McMahon (13) purified an aromatic aldehyde-ketone reductase from rabbit kidney, but did not examine its physical properties and it is not clear whether the enzyme was separated from aldehyde reductase or not. With respect to cofactor and substrate specificity, the guinea pig liver aromatic aldehyde-ketone reductases (AR 1 and AR 2) resemble the rabbit kidney enzyme. It
became clear that AR 1 and AR 2 were separated from aldehyde reductase (AR 3). Recently, two aldehyde reductase isozymes have been isolated from rat liver (6) and mouse liver (7), but AR 1 and AR 2 are different from all these isozymes, for which aromatic ketones are poor substrates. Liver alcohol dehydrogenase reduces aldehydes and cyclic ketones (1, 3). AR 1 and AR 2, however, are different from alcohol dehydrogenase in cofactor specificity and inhibitor sensitivity. Moreover, alcohol dehydrogenase activity was separated from the two reductases on this purification. AR 1 and AR 2 are apparently different from a,fiunsaturated ketone reductase of human liver and dog erythrocyte (14) because of their broad substrate specificity. In our recent work (16), two aromatic aldehyde-ketone reductases were isolated from rabbit liver, showing low molecular weights of 30,000 and similar substrate specificity to the rabbit kidney enzyme (13) and the guinea pig liver enzymes. However, these enzymes are different in inhibitor sensitivity; the enzymes in rabbit and guinea pig liver are not affected by metal binding reagents that inhibit the kidney enzyme, and one of the rabbit liver enzymes is more sensitive to />-chloromercuribenzoate that any of the guinea pig liver enzymes. In substrate specificity, moreover, the guinea pig enzymes showed lower Km values for /vnitroacetophenone than for benzoylpyridines, whereas the rabbit enzymes showed higher affinity for benzoylpyridines. AR 1 and AR 2 are markedly different in their relative maximal velocities and Km values for the various substrates examined, and in their inhibitor sensitivity and heat stability. Interconversion of the purified AR 1 and AR 2 was never detected. Similar heterogeneity was observed in kidney enzymes. In previous work (IS), the aromatic ketone reductase activity was found in both the cytosolic and microsomal fractions. Therefore, one of the two enzymes may not be a degradation artifact, but may be microsomal in origin. Both AR 1 and AR 2 were also able to catalyze the oxidoreduction between cyclohexanol and cyclohexanone, which are the simplest steroid analogues. As will be discussed in the following paper (29), the physiological function of these enzymes might be in steroid metabolism in addition to detoxication of xenobiotic aldehydes and ketones. / . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/86/4/871/770838 by Mount Sinai Icahn School of Medicine user on 03 February 2019
contaminant. Although they are similar in molecular weight, about 35,000, they are markedly different in other properties. A major difference of these enzymes is in substrate specificity; AR 1 and AR 2 reduced aromatic ketones as well as aromatic aldehydes, whereas AR 3 reduced aromatic and aliphatic aldehydes and some aldoses such as r>glucuronate but not aromatic ketones. Judging from catalytic and physical properties, AR 3 is apparently the aldehyde reductase which has been purified from the livers of several species, such as the rat (5, 6), mouse (7), rabbit {16), and man (8). The mouse liver enzyme is inactive towards aliphatic aldehydes and is weakly active on a number of sugars (7), but AR 3 reduced the former but not the latter like the enzymes from the other species. The physiological role of this enzyme has been considered to be in the reduction of D-glucuronate to L-gulonate for the production of L-ascorbic acid (4, 7). The question is whether aldehyde reductase plays this role, because the enzyme is present in man (8) and the guinea pig, two mammals known to be unable to synthesize L-ascorbic acid (27). Aldehyde reductase is widely distributed in tissues (6) and the enzymes purified from various tissues show similar properties to those of the liver enzyme (4, 6, 9, 28). The enzyme in bovine heart has a high affinity for fatty aldehydes and its role is in the synthesis of fatty acids (28). The brain enzyme plays a role in the metabolism of aldehydes derived by the deamination of biogenic amines (9). In this work, one of the aldehyde reductases in various tissues of the guinea pig was immunochemically identical to AR 3 from liver, and fatty aldehydes were poor substrates for AR 3. Therefore, the general role of this enzyme may be in detoxication of biogenic and xenobiotic aldehydes.
REDUCTASES FOR AROMATIC ALDEHYDES AND KETONES
REFERENCES 1. Merrit, A.D. & Tomkins, G.M. (1959) / . Biol. Chem. 234, 2778-2782 2. Reynier, M. & Theorell, H. (1969) Ada Chem. Scand.H, 1130-1136 3. Pietruszko, R., Crawford, K., & Lester, D. (1973) Arch. Biochem. Biophys. 159, 50-60 4. Bosron, W.F. & Prairie, R.L. (1972) / . Biol. Chem. 247, 4480-4485 5. Felsted, R.L., Richter, D.R., & Bachur, N.R. (1977) Biochem. Pharmac. 26, 1117-1124 6. Erwin, V.G. & Deitrich, R.A. (1972) Biochem. Pharmac. 21, 2915-2924 7. Tulsiani, D.R.P. & Touster, O. (1977) / . Biol. Chem. 252, 2545-2550 8. Wennuth, B., Munch, J.D.B., & von Wartburg, J.-P. (1977) / . Biol. Chem. 252, 3821-3828 9. Turner, A.J. & Tipton, K.F. (1972) Biochem. J. 130, 765-772 10. Ris, M.M. & von Wartburg, J.-P. (1973) Eur. J. Biochem. 37, 69-77
Vol. 86, No. 4, 1979
11. Bosron, W.F. & Prairie, R.L. (1973) Arch. Biochem. Biophys. 154, 166-172 12. Bachur, N.R. (1976) Science 193, 595-597 13. Culp, H.W. & McMahon, R.E. (1968) /. Biol. Chem. 243, 848-852 14. Fraser, I.M., Peters, M.A., & Hardinge, M.G. (1967) Mol. Pharmacol. 3, 233-247 15. Sawada, H. & Hara, A. (1978) Drug Metab. Dispos. 6,205-211 16. Sawada, H. & Hara, A. (1979) Biochem. Pharmac. 28, 1089-1094 17. Sawada, H., Sasaki, E., Asano, S., & Hara, A. (1978) Yakugaku Zasshi (in Japanese) 98,1167-1172 18. Bohme, H.-J., Kopperschlager, G., Schulz, J., & Hofmann, E. (1972) / . Chromatogr. 69, 209-214 19. Levine, 0 . (1962) in Methods in Enzymology (Colowick, S.P. & Kaplan, N.O., eds.) Vol. V, pp. 27-32, Academic Press, New York 20. Lowry, O.H., Rosebrough, NJ., Farr, A.L., & Randall, R.J. (1951) / . Biol. Chem. 193, 265-275 21. Davis, B.J. (1964) Ann. N.Y. Acad. Sci. Ill, 404-427 22. Weber, K. & Osborn, M. (1969) / . Biol. Chem. 244, 4406^412 23. Siegel, L.M. & Monty, K.J. (1966) Biochim. Biophys. Ada 112, 346-362 24. Laurent, T.C. & Killander, J. (1964) / . Chromatogr. 14, 317-330 25. Ouchterlony, O. (1949) Ada Pathol. Microbiol. Scand. 16, 507-515 26. Eckfeldt, J., Mope, L., Takio, K., & Yonetani, T. (1976) / . Biol. Chem. 251, 236-240 27. Burns, J J. (1960) in Metabolic Pathways (Greenberg, D.M., ed.) Vol. 1, pp. 341-346, Academic Press, New York 28. Kawaled, J.C. & Gilbertson, J.R. (1976) Arch. Biochem. Biophys. 173, 649-657 29. Sawada, H., Hara, A., Hayashibara, M., & Nakayama, T. (1979) / . Biochem. 86, 883-892
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Aldehyde reductase isozymes, which have the} ability to reduce p-nitroacetophenone and benzylmethylketone, have been isolated from human and rat brain (70). In the experiments on the tissue distribution of reductases in the guinea pig, AR 1 and AR 2 were chromatographically found to be only in liver and kidney. Other tissues may contain only AR 3, or the activity of the other two enzymes in other tissues may be too little to detect on chromatography. However, lung contained another ketone reductase different from AR 1 and AR 2. The presence of such a high molecular weight aromatic ketone reductase has apparently not been reported before.
881
and Ketones from Guinea Pig Liver Hideo SAW ADA, Akira HARA, Fumiko KATO, and Toshihiro NAKAYAMA Gifu College of Pharmacy, Mitahora-higashi, Gifu, Gifu 502 Received for publication, February 10, 1979
NADPH-dependent enzymatic reduction of aromatic aldehydes and ketones observed in the cytosol of guinea pig liver was mediated by at least three distinct reductases (AR 1, AR 2, and AR 3), which were separated by DEAE-cellulose chromatography. By several procedures AR 2 and AR 3 were purified to homogeneity, but AR 1 could be purified only 30-fold because of the small amount. These enzymes were found to have similar molecular weights of 34,000 to 36,000 and similar Stokes radii of about 2.5 nm. AR 3 was identical to aldehyde reductase [EC 1.1.1.2] in substrate specificity for aromatic aldehydes and Dglucuronate and specific inhibition by barbiturates. AR 1 and AR 2 acted on aromatic ketones and cyclohexanone as well as aromatic aldehydes at optimal pHs of 5.4 and 6.0, respectively, and were immunochemically distinguished from AR 3. AR 1 was the most sensitive to sulfhydryl reagents, and AR 2 was more stable at 50°C than the other enzymes. Similar heterogeneity was observed in the kidney enzymes, but other tissues had little aldehyde reductase activity and contained only AR 3. In addition, lung contained a high molecular weight aromatic ketone reductase different from the above reductases.
Biological reduction of biogenic and xenobiotic carbonyl compounds to their corresponding alcohols is mediated by several cytoplasmic pyridine nucleotide-dependent oxidoreductases. Alcohol dehydrogenase catalyzes the reduction of aldehydes, alicyclic ketones, and some steroids in the presence of NADH (1-3). NADPH-dependent aldehyde reductase catalyzes the reduction of aldehydes and some aldoses, but is generally inactive towards ketones (4-10). However, aldehyde reductases differing in substrate specificity have been isolated from several mammalian species. The pig kidney Abbreviations: AR 1, aldehyde reductase 1; AR 2, aldehyde reductase 2; AR 3, aldehyde reductase 3. Vol. 86, No. 4, 1979
enzyme is active on a-diketones (4), whereas the enzymes from other animal tissues reduce only aldehydes (5-9). Four multiple molecular forms of this enzyme have been isolated from human brain and two of them have the ability to reduce several aromatic ketones (10). In addition, a high molecular weight aldehyde reductase with high reactivity for cyclohexanone and D-ribose has been observed in several mammalian livers (11). On the other hand, the enzymatic reduction of a number of ketones has been reviewed (12), but the reductases responsible for this have rarely been purified except for the reductase for aromatic aldehydes and ketones from rabbit kidney (13) and a,/9-unsaturated ketone reductase from human liver and dog
871
Downloaded from https://academic.oup.com/jb/article-abstract/86/4/871/770838 by Mount Sinai Icahn School of Medicine user on 03 February 2019
Purification and Properties of Reductases for Aromatic Aldehydes
H. SAWADA, A. HARA, F. KATO, and T. NAKAYAMA
872
EXPERIMENTAL PROCEDURE Materials—All chemicals were of reagent grade and were purchased from commercial sources. p-Nitrobenzyl alcohol and p-nitroacetophenone were recrystallized from ethanol. Phenyl-(jpyridyl)carbinol was synthesized from 4-benzoylpyridine by addition of an excess of NaBH4 in methanol and recrystallized from chloroform. Proteins used for Sephadex column and polyacrylamide gel calibration were purchased from Sigma Chemical Co. and Seikagaku Kogyo Co., respectively. Human prostatic acid phosphatase was isolated by the method reported previously (17). Sephadex G-75 and G-100 were obtained from Pharmacia Fine Chemicals and DEAE-cellulose (DE32) from Whatman. Cibacron-substituted Sephadex and hydroxylapatite were prepared by the methods of Bohme et al. (18) and Levine (19), respectively. Buffers—The following buffers were used. Buffer A, 10 mM Tris-HCl buffer containing 5 mM 2-mercaptoethanol and 0.5 mM EDTA, pH7.5; Buffer B, 10 mM Tris-HCl buffer containing 5 mM 2-mercaptoethanol and 1 mM K2HPO4, pH7.5; Buffer C, 50 mM Tris-HCl buffer containing 1 mM 2-mercaptoethanol and 0.15 M KC1, pH 7.5; Buffer D, 50 mM Tris-HCl buffer (pH 8.5), 5 mM 2mercaptoethanol, 0.5 mM EDTA, and 0.6 M ammonium sulfate (adjusted to pH 8.5 with 28 % NH4OH). All pH titrations were performed at 25°C. Enzyme Assay—Enzyme activity was measured by the rate of NADPH oxidation monitored at 340 nm. Unless otherwise specified, the assay
mixture contained, in a total volume of 2.5 ml, 80 mM phosphate buffer (pH 6.0) or citrate buffer (pH 5.4), 0.08 mM NADPH, an appropriate amount of enzyme preparation, and 2.5 / glucuronate and p-nitroacetophenone as substrates. In gel filtration both activities of all the tissues except for lung gave a single broad peak at a similar low molecular weight of about 34,000, but it was found that, in addition to the low molecular weight active peak, lung also contained a high molecular weight active peak that had only a high p-nitroacetophenone reductase activity. Figure 7 shows elution profiles of the reductase activities of the low and high molecular weight fractions from each tissue on DE32 chromatography. Activities of aldehyde reductases in kidney showed a similar elution pattern to that in liver (Fig. 1), but the low molecular weight reductases of lung, brain, and heart (data not shown) gave a single active peak that corresponded to liver AR 3. The high molecular weight reductase of lung was eluted at a
Vol. 86, No. 4, 1979
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Fig. 8. Immunological characterization of aldehyde reductases by the Ouchterlony double-diffusion technique. Plate A, center well contained anti-AR 3 serum, peripheral wells contained the following; (1), AR 3; (2), AR 2; (3), AR 1; (4), enzyme preparation from the Sephadex G-100 step. Plate B, center well contained anti-AR 3 serum, peripheral wells contained AR 3-fractions of DE32 chromatography from lung (1), kidney (2), heart (3), and brain (4).
different NaCl concentration from the other reductases. Double Diffusion Gel Precipitations—Antisera against AR 3 from guinea pig liver were produced in rabbits. By use of the Ouchterlony double diffusion test (Fig. 8A), the antisera gave a single and sharp precipitin line against the antigens and the liver enzyme preparation from the Sephadex G-100 step, but no precipitin line was observed with AR 1 and AR 2. Immunodiffusion against the AR 3-fractions obtained from DE32 chromatography of enzymes from different tissues of the same species resulted in a single precipitin line with the antisera against liver AR 3, and the lines fused completely with each other (Fig. 8B). DISCUSSION We have demonstrated the existence in guinea pig liver of at least three distinct NADPH-dependent enzymes which catalyze the reduction of aldehydes to alcohols, and have presented procedures by which the three reductases (AR 1, AR 2, and AR 3) can be isolated from the cytosol of guinea pig liver. Our AR 2 and AR 3 preparations were judged to be pure by polyacrylamide disc gel electrophoresis with and without sodium dodecyl sulfate and an agarose diffusion test with rabbit anti-AR 3 serum, but AR 1 contained a minor
880
H. SAWADA, A. HARA, F. KATO, and T. NAKAYAMA
In spite of many reports on enzyme activities reducing aromatic aldehydes and ketones, as reviewed by Bachur (12), the reductases responsible for ketones have not been purified sufficiently yet. Culp and McMahon (13) purified an aromatic aldehyde-ketone reductase from rabbit kidney, but did not examine its physical properties and it is not clear whether the enzyme was separated from aldehyde reductase or not. With respect to cofactor and substrate specificity, the guinea pig liver aromatic aldehyde-ketone reductases (AR 1 and AR 2) resemble the rabbit kidney enzyme. It
became clear that AR 1 and AR 2 were separated from aldehyde reductase (AR 3). Recently, two aldehyde reductase isozymes have been isolated from rat liver (6) and mouse liver (7), but AR 1 and AR 2 are different from all these isozymes, for which aromatic ketones are poor substrates. Liver alcohol dehydrogenase reduces aldehydes and cyclic ketones (1, 3). AR 1 and AR 2, however, are different from alcohol dehydrogenase in cofactor specificity and inhibitor sensitivity. Moreover, alcohol dehydrogenase activity was separated from the two reductases on this purification. AR 1 and AR 2 are apparently different from a,fiunsaturated ketone reductase of human liver and dog erythrocyte (14) because of their broad substrate specificity. In our recent work (16), two aromatic aldehyde-ketone reductases were isolated from rabbit liver, showing low molecular weights of 30,000 and similar substrate specificity to the rabbit kidney enzyme (13) and the guinea pig liver enzymes. However, these enzymes are different in inhibitor sensitivity; the enzymes in rabbit and guinea pig liver are not affected by metal binding reagents that inhibit the kidney enzyme, and one of the rabbit liver enzymes is more sensitive to />-chloromercuribenzoate that any of the guinea pig liver enzymes. In substrate specificity, moreover, the guinea pig enzymes showed lower Km values for /vnitroacetophenone than for benzoylpyridines, whereas the rabbit enzymes showed higher affinity for benzoylpyridines. AR 1 and AR 2 are markedly different in their relative maximal velocities and Km values for the various substrates examined, and in their inhibitor sensitivity and heat stability. Interconversion of the purified AR 1 and AR 2 was never detected. Similar heterogeneity was observed in kidney enzymes. In previous work (IS), the aromatic ketone reductase activity was found in both the cytosolic and microsomal fractions. Therefore, one of the two enzymes may not be a degradation artifact, but may be microsomal in origin. Both AR 1 and AR 2 were also able to catalyze the oxidoreduction between cyclohexanol and cyclohexanone, which are the simplest steroid analogues. As will be discussed in the following paper (29), the physiological function of these enzymes might be in steroid metabolism in addition to detoxication of xenobiotic aldehydes and ketones. / . Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/86/4/871/770838 by Mount Sinai Icahn School of Medicine user on 03 February 2019
contaminant. Although they are similar in molecular weight, about 35,000, they are markedly different in other properties. A major difference of these enzymes is in substrate specificity; AR 1 and AR 2 reduced aromatic ketones as well as aromatic aldehydes, whereas AR 3 reduced aromatic and aliphatic aldehydes and some aldoses such as r>glucuronate but not aromatic ketones. Judging from catalytic and physical properties, AR 3 is apparently the aldehyde reductase which has been purified from the livers of several species, such as the rat (5, 6), mouse (7), rabbit {16), and man (8). The mouse liver enzyme is inactive towards aliphatic aldehydes and is weakly active on a number of sugars (7), but AR 3 reduced the former but not the latter like the enzymes from the other species. The physiological role of this enzyme has been considered to be in the reduction of D-glucuronate to L-gulonate for the production of L-ascorbic acid (4, 7). The question is whether aldehyde reductase plays this role, because the enzyme is present in man (8) and the guinea pig, two mammals known to be unable to synthesize L-ascorbic acid (27). Aldehyde reductase is widely distributed in tissues (6) and the enzymes purified from various tissues show similar properties to those of the liver enzyme (4, 6, 9, 28). The enzyme in bovine heart has a high affinity for fatty aldehydes and its role is in the synthesis of fatty acids (28). The brain enzyme plays a role in the metabolism of aldehydes derived by the deamination of biogenic amines (9). In this work, one of the aldehyde reductases in various tissues of the guinea pig was immunochemically identical to AR 3 from liver, and fatty aldehydes were poor substrates for AR 3. Therefore, the general role of this enzyme may be in detoxication of biogenic and xenobiotic aldehydes.
REDUCTASES FOR AROMATIC ALDEHYDES AND KETONES
REFERENCES 1. Merrit, A.D. & Tomkins, G.M. (1959) / . Biol. Chem. 234, 2778-2782 2. Reynier, M. & Theorell, H. (1969) Ada Chem. Scand.H, 1130-1136 3. Pietruszko, R., Crawford, K., & Lester, D. (1973) Arch. Biochem. Biophys. 159, 50-60 4. Bosron, W.F. & Prairie, R.L. (1972) / . Biol. Chem. 247, 4480-4485 5. Felsted, R.L., Richter, D.R., & Bachur, N.R. (1977) Biochem. Pharmac. 26, 1117-1124 6. Erwin, V.G. & Deitrich, R.A. (1972) Biochem. Pharmac. 21, 2915-2924 7. Tulsiani, D.R.P. & Touster, O. (1977) / . Biol. Chem. 252, 2545-2550 8. Wennuth, B., Munch, J.D.B., & von Wartburg, J.-P. (1977) / . Biol. Chem. 252, 3821-3828 9. Turner, A.J. & Tipton, K.F. (1972) Biochem. J. 130, 765-772 10. Ris, M.M. & von Wartburg, J.-P. (1973) Eur. J. Biochem. 37, 69-77
Vol. 86, No. 4, 1979
11. Bosron, W.F. & Prairie, R.L. (1973) Arch. Biochem. Biophys. 154, 166-172 12. Bachur, N.R. (1976) Science 193, 595-597 13. Culp, H.W. & McMahon, R.E. (1968) /. Biol. Chem. 243, 848-852 14. Fraser, I.M., Peters, M.A., & Hardinge, M.G. (1967) Mol. Pharmacol. 3, 233-247 15. Sawada, H. & Hara, A. (1978) Drug Metab. Dispos. 6,205-211 16. Sawada, H. & Hara, A. (1979) Biochem. Pharmac. 28, 1089-1094 17. Sawada, H., Sasaki, E., Asano, S., & Hara, A. (1978) Yakugaku Zasshi (in Japanese) 98,1167-1172 18. Bohme, H.-J., Kopperschlager, G., Schulz, J., & Hofmann, E. (1972) / . Chromatogr. 69, 209-214 19. Levine, 0 . (1962) in Methods in Enzymology (Colowick, S.P. & Kaplan, N.O., eds.) Vol. V, pp. 27-32, Academic Press, New York 20. Lowry, O.H., Rosebrough, NJ., Farr, A.L., & Randall, R.J. (1951) / . Biol. Chem. 193, 265-275 21. Davis, B.J. (1964) Ann. N.Y. Acad. Sci. Ill, 404-427 22. Weber, K. & Osborn, M. (1969) / . Biol. Chem. 244, 4406^412 23. Siegel, L.M. & Monty, K.J. (1966) Biochim. Biophys. Ada 112, 346-362 24. Laurent, T.C. & Killander, J. (1964) / . Chromatogr. 14, 317-330 25. Ouchterlony, O. (1949) Ada Pathol. Microbiol. Scand. 16, 507-515 26. Eckfeldt, J., Mope, L., Takio, K., & Yonetani, T. (1976) / . Biol. Chem. 251, 236-240 27. Burns, J J. (1960) in Metabolic Pathways (Greenberg, D.M., ed.) Vol. 1, pp. 341-346, Academic Press, New York 28. Kawaled, J.C. & Gilbertson, J.R. (1976) Arch. Biochem. Biophys. 173, 649-657 29. Sawada, H., Hara, A., Hayashibara, M., & Nakayama, T. (1979) / . Biochem. 86, 883-892
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Aldehyde reductase isozymes, which have the} ability to reduce p-nitroacetophenone and benzylmethylketone, have been isolated from human and rat brain (70). In the experiments on the tissue distribution of reductases in the guinea pig, AR 1 and AR 2 were chromatographically found to be only in liver and kidney. Other tissues may contain only AR 3, or the activity of the other two enzymes in other tissues may be too little to detect on chromatography. However, lung contained another ketone reductase different from AR 1 and AR 2. The presence of such a high molecular weight aromatic ketone reductase has apparently not been reported before.
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