Xenobiotica the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Purification and molecular properties of 2carboxybenzaldehyde (CBA) reductase from phenobarbital-treated rat liver K. Tonda & M. Hirata To cite this article: K. Tonda & M. Hirata (1992) Purification and molecular properties of 2carboxybenzaldehyde (CBA) reductase from phenobarbital-treated rat liver, Xenobiotica, 22:6, 691-699, DOI: 10.3109/00498259209053131 To link to this article: http://dx.doi.org/10.3109/00498259209053131
Published online: 22 Sep 2008.
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Date: 06 April 2016, At: 12:56
XENOBIOTICA,
1992, VOL. 22,
NO.
6, 691-699
Purification and molecular properties of 2-carboxybenzaldehyde (CBA) reductase from phenobarbital-treated rat liver K. T O N D A and M. HIRATA" Shionogi Research Laboratories, Shionogi & Co., Ltd, Fukushima-ku, Osaka 553, Japan
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Received 5 November 1991; accepted 20 March 1992
1. A rat liver cytosol enzyme, tentatively named CBA reductase, catalyses the conversion of 2-carboxybenzaldehyde (CBA) to 2-hydroxymethyl benzoic acid in the presence of NADH (or NADPH). CBA reductase is useful for exploring the mechanism of in vitro enzyme induction, as the enzyme can be induced by phenobarbital (PB) both in vivo and in vitro. 2. Possible involvement of glutathione (GSH) in gene expression was suggested by a recent study with cultured rat hepatocytes. 3. CBA reductase was purified about 200-fold by a combination of column chromatography and isoelectric focusing in the presence of mercaptoethanol.
4. The ability to form 2-hydroxymethyl benzoic acid was lost when the enzyme was chromatographed on a hydroxylapatite column in the absence of mercaptoethanol; however, it was restored if sulphydryl compounds or bovine serum albumin was added to the eluate from the column.
5. Gel filtration showed the molecular sizes of CBA reductase from the lO5OOOg supernatant fraction of rat liver extracts and the purified preparation were 64 kDa and 49 kDa, respectively. 6. The results suggest that sulphydryl substances and some proteins play important roles in preserving the molecular and catalytic properties of CBA reductase.
Introduction We have previously shown that a rat liver enzyme in the cytosol catalyses the reduction of 2-carboxybenzaldehyde (CBA) to 2-hydroxymethyl benzoic acid (HMB) in the presence of NADH (Tonda et al. 1987). 3- or 4-Carboxybenzaldehyde could not serve as substrate of the enzyme (Tonda and Hirata 1987), although they are oxidized to their corresponding acids in rat liver (Shiobara and Ogiso 1979). T h e enzyme, tentatively named CBA reductase, was induced by phenobarbital (PB) both in vivo and in vitro (Hirata et al. 1986). CBA reductase activity can be determined either by a spectrophotometric method, which measures the substrate-dependent consumption of NADH, or by h.p.l.c., which quantifies the amount of HMB formed. T h e catalytic activity of the enzyme was abolished when treated with sulphydryl reagents, while 10 mM pyrazole, an alcohol dehydrogenase inhibitor (Branden et al. 1975), or 1 mM barbital, an aldehyde reductase inhibitor (Ahmed et al. 1979), had no effect at all. Since the substrate specificity and responses to various inhibitors differed from those of known dehydrogenases, we attempted to purify the enzyme from liver extracts of PB-treated rat, by a combination of gel filtration, hydrophobic interaction, hydroxylapatite column chromatography and isoelectric focusing. Hydroxylapatite treatment disclosed unique molecular properties of CBA reductase. This report describes the purification of CBA reductase from PB-treated rat liver and various factors regulating the enzyme activity.
* T o whom correspondence should be addressed. 0049%3254/92 $3.00 0 1992 Taylor & Francis Ltd
692
K . Tonda and M . Hirata
Materials and methods Chemicals Phenobarbital-sodium (PB), 2-carboxybenzaldehyde (CBA), hydroxymethylbenzoic acid (HMB), 2mercaptoethanol and hydroxylapatite (100-350 mesh) were purchased from Nacalai Tesque Inc. (Kyoto, Japan), p-Chloromercuribenzoic acid (p-CMB), N-ethylmaleimide, pyridoxal-5-phosphate, NADH and NADPH from Sigma; dithiothreitol and glyoxalic acid from Wako Pure Chem. (Osaka, Japan); 2formylphenoxyacetic acid, Sephadex G-150 superfine and Phenyl Sepharose CL-4B from Pharmacia (Uppsala, Sweden); PIC reagent (Low UVA) was obtained from Waters Assoc. Inc. (Milford, MA) and Toyopearl HW-50 from Toyo Soda Inc. (Tokyo, Japan). Other chemicals were of reagent grade.
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Animals Male Wistar rats, 10-11 weeks old and weighing 300-3309, were used. The rats were treated with PB (80mg/kg per day) in saline injected i.p. for 3 days, and were killed 24h after the last injection. Preparation of liver extracts The rat liver was excised, after perfusion with saline under ether anaesthesia, and homogenized with 5 vol. of 50mM phosphate buffer, pH 7.4, containing 3 mM mercaptoethanol by Potter homogenizer or Ultra-turrax (TKA-Werk). The homogenate was centrifuged first for 2 min at 250g then at 11 OOOg for lOmin at 4°C. The supernatant was further centrifuged at lO5OOOg for 80min at 4°C. Purification of CBA reductase Ammonium sulphate fractionation. Ammonium sulphate was added to the 105 OOOg supernatant at 4°C and the precipitate at 3&50% saturation was collected by centrifugation at 7000g for 10min. T h e precipitate was dissolved in 50mM phosphate buffer with 3 mM mercaptoethanol, pH 7.0, and dialysed overnight at 4°C against the same buffer. Gel chromatography. T h e dialysed enzyme preparation was concentrated by centrifugation with an Amicon Centriflo filter (CF-25) at 450g, and a 6-ml portion was chromatographed on a Toyopearl TSKGel HW 50 column ( 2 6 x 65 cm) with buffer A (50mM phosphate buffer, pH 6.8, containing 3 mM mercaptoethanol). CBA reductase activity in each 5 ml fraction was immediately assayed. Phenyl Sepharose column chromatography. The active fraction from gel chromatography was diluted 5fold with distilled water and applied to a Phenyl Sepharose CL-4B column (2.2 x 15 cm), which had been equilibrated with 5-fold diluted buffer A. After washing the column with buffer A, the enzyme was eluted into lOml tubes with 50% propylene glycol containing 3 mM mercaptoethanol. The eluate was dialysed overnight at 4°C against buffer B (1OmM phosphate buffer-3 mM mercaptoethanol, p H 70). Hydroxylapatite column chromatography. The above enzyme preparation was adsorbed onto a hydroxylapatite column (2.2 x 5.5 cm) which had been equilibrated with buffer B, and washed with the buffer. CBA reductase was then eluted with 38 mM phosphate buffer containing 3 mM mercaptoethanol, pH 6.8. Zsoelectric focusing. The active fraction obtained from hydroxylapatite chromatography was concentrated 5-fold using Amicon Centriflo CF-25, and applied to an LKB isoelectric focusing column (1 10 cm) at 4°C with sucrose density gradient in 0.05% Ampholine (pH 3-10). Electrophoresis was conducted for 20 h at 500 V and 5 mA, then for 2 h at 900 V and 1 mA in the presence of 3 mM mercaptoethanol at 4°C. After fractionation into 1-ml portions, the p H value and U.V.absorbance at 280nm of the fraction was determined. The fraction was dialysed before protein determination. Determination of CBA reductase activity CBA reductase was assayed both by spectrophotometry and h.p.1.c.; the former measures the substrate-dependent consumption of NADH and the latter the formation of HMB from CBA. Spectrophotometry. A portion of the enzyme preparation was preincubated with NADH ( 1 6 0 ~ in ~) 50mM phosphate buffer, pH 7.0, at 37"C, and the reaction was initiated by adding CBA (1 mM, final). The absorbance at 340nm was monitored with a Shimadzu UV 300 spectrophotometer. H.p.1.c. A portion of the enzyme preparation was preincubated with NADH (2 mM) in 50 mM phosphate buffer, pH 7.0, for 5 min at 37"C, then CBA (1 mM) was added. After 10min of reaction at 37"C, a 1-ml portion was transferred to a tube with 0.25 ml 0 4 M perchloric acid containing 125 pg sulphanilamide (internal standard for h.p.1.c.) and 2ml ethyl acetate. The tube was vigorously shaken for lOmin, centrifuged for 5 min at 700g,then a 300 p! portion of the organic layer was evaporated in uucuo, and dissolved in the h.p.1.c. mobile phase (600~1)supplemented with lop1 M-NaOH. H.p.1.c. was conducted on a Cosmosil 5 Ph column (Nacalai Tesque Inc., 4.6 x 1SOmrn), with water-acetonitrile (10: 1 v/v)/Pic reagent-A (low u.v.) (98 : 2 v/v) as the mobile phase and detected at 230 nm by a Shimadzu SCL-6A liquid chromatography system at 25°C. The retention times for CBA, HMB and sulphanilamide were 560,480 and 290 s, respectively, at a flow rate of 1 ml/min. Protein was determined according to Lowry et al. (1951)
693
Purification of carboxybenzaldehyde reductase
using bovine serum albumin as the standard. T h e protein concentration of the eluate from the isoelectric focusing system was assayed by measuring the ratio of absorbance at 260 and 280nm as described by Dawson et al. (1972).
Results Purification of CBA reductase
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CBA reductase was purified about 200-fold from the 105 OOOg supernatant fraction of liver extracts of PB-treated rats, by ammonium sulphate fractionation, Toyopearl-HW 50 gel filtration, Phenyl Sepharose CL-4B hydrophobic chromatography, hydroxylapatite chromatography and isoelectric focusing in the presence of 3 mM mercaptoethanol (table 1). T h e overall yield of enzyme activity was 8% and the isoelectric point (Pi)of the purified enzyme was 6.96 (figure 1). T h e apparent K , value for CBA was 1.7 mM at p H 7.0 with 2 mM NADH and 3 mM mercaptoethanol, Table 1. Purification of 2-carboxybenzaldehyde (CAB) reductase from PB-treated rat liver. Total protein (md
Steps of purification 105 OOOg Supernatant Ammonium sulphate (3@500/,) Toyopearl IW-50 Phenyl Sepharose CL-4B Hydroxylapatite Isoelectric focusing
Total activity' (pmol/min)
5817 2167 1397 257 37 2.Sb
Specific activity Yield (pmol/min per mg) (%) Purification
307 309 251 147 95 26
100 101 82 48 31 8
005 0.14 018 0.57 2.58 1039
1.o 2.7 3.4 10.0 48.9 197.0
"CBA reductase activity was determined in the presence of 3 mM mercaptoethanol (h.p.1.c. method). Protein concentration was calculated by the equation based on the extinction at 260 nm and 280 nm according to Dawson et al. (1972).
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Figure 1. Isoelectric focusing profile of 2-carboxybenzaldehyde (CBA) reductase obtained from hydroxylapatite column chromatography. Fractions (1 ml/tube) were analysed for CBA reductase and protein by measuring the NADH oxidation and U.V.absorbance at 280 nm, respectively.
K . Tonda and M . Hirata
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694
3 4 5 2-Carboxybenzaldehyde [ 1 / S : mM-’] 1
2
Figure 2. Enzyme kinetics of 2-carboxybenzaldehyde (CBA) reductase as a function of (A) CBA ~ ~ and (B)NADH concentration in the presence of concentration in the presence of 1 6 0 NADH 2mM CBA. Enzyme purified from isoelectric focusing was used, and the enzyme activity was assayed with 3 mM mercaptoethanol (h.p.1.c. method). For CBA: K,= 1.68 mM, VmaX =27.6 pnol/min per mg protein. For NADH: K,,,= 0.50 mM, VmaX = 12.7pmol/min per mg protein.
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Hydroxylapatite column chromatography profile of 2-carboxybenzaldehyde (CBA) reductase obtained from Phenyl Sepharose CL-4B hydrophobic chromatography. The enzyme was purified without mercaptoethanol, and the activity was determined in the presence or absence of mercaptoethanol.
Figure 3.
Purification of carboxybenzaldehyde reductase Table 2.
Effect of sulphydryl compounds and proteins on 2-carboxybenzaldehyde (CBA) reductase obtained from hydroxylapatite column chromatography. Experiment 1 Additive
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Experiment 2
Activity (percentage of control)
Additive
Activity (percentage of control)
0
None 3 mM 1 mM 3 mM 3 mM
695
Mercaptoethanol Dithiothreitol CSH (reduced) GSH (oxidized)
3 mM S-Methyl-GSH 3 mM Cysteine 3 mM Cystine 3 mM Methionine
100" 103 103
81 41 100 69 62
Bovin serum albumin (O.Olb) Bovin serum albumin (0.05) Bovin serum albumin (0.10) Bovin serum albumin (0.50) Bovin serum albumin (0.50, boiled for 10 min) Ovalbumin (0.50) y-Globulin (0.50) 3 mM Mercaptoethanol
2 92 105 106 108
0 15 100'
CBA reductase activity was determined in the absence of mercaptoethanol (h.p.1.c.method). 2.39 pmol/min per mg protein. 'mg/rnl in the reaction mixture. '5.31 pmol/min per mg protein. a
while the K,,, value for NADH with 1 mM CBA was 0-5mM (figure 2). In a previous study the K,,, value in the 105 OOOg supernatant fraction for CBA was found to be 1 mM (Hirata et al. 1986).
Substrate specificity T h e catalytic activity of the enzyme appears to be specific for CBA. T h e activity toward 3- or 4-carboxybenzaldehyde was negligibly small, as described previously (Tonda and Hirata 1987). Pyrazole and o-phenanthroline, which inhibit alcohol dehydrogenase, or barbital, an inhibitor of aldehyde reductase, did not inhibit CBA reductase. Variation of N A D H concentration may profoundly influence the enzyme activity as it was increased by 225% (1.6-5.2pmol/min per mg protein) when the concentration of NADH increased from 0.2 to 2 . 0 m ~though , it was increased by only 20% (1.1-1.4pmol/min per mg protein) with increased concentrations of NADPH (0-2-2.0m~).T h e effect of molecular oxygen on CBA reductase was examined using a glucose oxidase-catalase system to deplete oxygen from the reaction mixture (Englander et al. 1987), but the treatment did not affect the enzyme activity at all. Inactivation and reactivation of the enzyme When CBA reductase was purified on a hydroxylapatite column without mercaptoethanol, the enzyme activity capable of producing HMB from CBA was completely lost (0.1 1-+O*OO), though CBA-dependent oxidation of N A D H (0.09-+0*58) took place (figure 3). T h e activity was, however, recovered (0-0.49 pmol HMB/min per ml) when a minute amount of 105 OOOg supernatant was added to the enzyme assay reaction mixture. Treatment of the supernatant with pronase (200 pg/ml) completely abolished the activity, while sulphydryl compounds, G S H , mercaptoethanol, dithiothreitol and cysteine, restored CBA reductase activity (table 2). Although less active, oxidized GSH, methionine and S-methyl G S H also reactivated the enzyme. It is particularly interesting that 0.005% bovine serum
K . Tonda and M . Hirata
696
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i I tubu number
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2 4 6 10 Log. M.W. ( ~ 1 0 4 )
Figure 4. Molecular weight estimation of 2-carboxybenzaldehyde (CBA) reductase obtained from hydroxylapatite column chromatography. The eluate from the column was 5-fold concentrated by centrifugation with Centriflo CF-25 (Amicon Corp') and a 1-ml portion was subjected to chromatography on a Sephadex (3-150 column (1.4 x 45 cm) CBA reductase activity was determined with 3 mM mercaptoethanol. One typical result of three experiments is presented. BSA = bovine serum albumin, or-Chymo = achymotrypsin, Ova = ovalbumin.
albumin reactivated CBA reductase, and furthermore the heat-treated albumin, 10min at 100°C, was still active. Ovalbumin did not reactivate the enzyme. Reactivated CBA reductase produced HMB with consumption of an equimolar amount of NADH. Molecular size of CBA reductase T h e molecular weight of CBA reductase was examined by gel filtration. T h e enzyme fraction eluted from the hydroxylapatite column was concentrated by Centriflo CF-25 (Amicon) and applied on a Sephadex G-150 column (1.4 x 45 cm). T h e CBA reductase activity was determined in the presence of 3 mM mercaptoethanol. T h e molecular weight of purified enzyme was estimated to be 49 kDa (figure 4). However, when the lO5OOOg supernatant of liver extracts was applied to the column, the activity was detected in the fraction corresponded to 64 kDa. This result suggests that CBA reductase could be dissociated to its subunits, or that the shape of the enzyme molecule markedly changed during the chromatography on hydroxylapatite column. Inhibitors of CBA reductase The effects of various aldehydes on purified CAB reductase were examined. They included glyoxylic acid, pyridoxal- 5-phosphate, glyceraldehyde-3-phosphate, oxaloacetic acid, pyruvic acid, dihydroacetone, benzaldehyde, 2-formylphenoxy acetic acid and p-nitrobenzaldehyde. Though they inhibited CBA reductase in the 105 OOOg supernatant, they were ineffective against the purified enzyme except that pyridoxal phosphate, benzaldehyde and formylphenoxy acetic acid inhibited the enzyme in a non-competitive manner. T h e apparent K ivalues for pyridoxal phosphate and formylphenoxy acetic acid were 0.75 and 0.58 mM, respectively
697
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Purification of carboxybenzaldehyde reductase
1
0
2
2-Carboxybenzaldehyde [ 1 / S : mM-’]
(A)
4
2 mM
>
1 2
1 rnM
F
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(B) Figure 5.
Effect of (A) 2-formylphenoxy acetic (2-FDA) and (B) pyridoxal-5-phosphate (PALP) on 2-carboxybenzaldehyde (CBA) reductase.
The enzyme preparation obtained from hydroxylapatite column chromatography and isoelectric focusing was used for (A) and (B), respectively. The enzyme activity was determined with 3 m M mercaptoethanol (h.p.1.c. method). V: pmol HMB formed/min per mg protein.
698
K. Tonda and M . Hirata
(figure 5 ) . They did not serve as the substrate for CBA reductase. Divalent metals interfere with the enzyme; C u z + at 1 mM inhibited the enzyme 75% and, Zn2+ and M o Z + about 30%, but EDTA or EGTA (1 mM) did not affect the activity significantly. In the absence of mercaptoethanol the enzyme was completely inhibited by p-CMB and N-ethylmaleimide. Though pyrazole, an alcohol dehydrogenase inhibitor, at 10 mM caused no inhibition at all, 1mM o-phenanthroline, another inhibitor, caused as much as 70% inhibition of the purified CBA reductase.
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Discussion Aromatic aldehydes are generally oxidized to their corresponding acids in animal tissues (Shiobara 1977). Unlike others, 2-carboxybenzaldehyde is reduced to its alcohol, and, despite the enzyme being a cytosolic one, it is inducible by phenobarbital (Hirata et al. 1986). T h e involvement of G S H in the enzyme induction was recently suggested, using primary culture of rat hepatocytes (Tonda and Hirata 1991). T o examine further the mechanism of induction, elucidation of the biochemical properties of CBA reductase would seem to be of value. T h e purified enzyme showed several characteristics.
1. T h e enzyme became physically unstable; for example, though the liver extracts passed through a Sephadex G-150 column and stored at -20°C retained their initial activity for over 2 months, the purified one lost 20% of its activity in 7 days and more than 60% after 14 days. 2. T h e apparent K , value increased from 1 mM to 1 . 7 m by ~ purification. 3. Molecular size of CBA reductase, as judged by gel filtration, in the 105 OOOg supernatant was estimated to be 64 kDa while that of the purified enzyme was 49 kDa. 4. Conversion of CBA to HMB was not observed with the purified enzyme, although the substrate-dependent oxidation of NADH took place. 5 . HMB-forming activity was recovered when the purified enzyme was treated with sulphydryl compounds including G S H , oxidized G S H , S-methyl G S H and methionine. 6 . Reactivation was also attained by bovine serum albumin, though ovalbumin was not effective. Thus, our results indicate that some regulatory subunit, which probably maintains thiol functions, could be dissociated during the enzyme purification. Sulphydryl substances may support the charge transfer from the NADH-reduced enzyme to the substrate CBA. A steroid 21 -hydroxylase purified from bovine adrenal glands showed similar catalytic properties in that the activity was restored by thiol compounds and bovine serum albumin (Greenfield et al. 1980), but subcellular distribution of the enzyme differs from that of CBA reductase. Steroid 21hydroxylase is integrated in microsomes of the tissue and its activity is NADPHdependent, while CBA reductase resides in the cytosol and NADH is preferentially used for its activity. T h e physiological significance of CBA reductase is not clear, but since endogenous substances so far examined could not serve as its substrate, the enzyme may be responsible for the metabolism of xenobiotics. The in vivo metabolism of formylbenzoic acids is particularly interesting in view of the regioselectivity exhibited. As mentioned above, CBA, which bears a carboxylic acid moiety in the ortho position, was exclusively reduced to its alcohol
Purification of carboxybenzaldehyde reductase
699
(HMB) in mammalian tissues and then excreted, while meta- and para-substituted analogues were oxidized to their corresponding dicarboxylic acids. Intramolecular hydrogen bond formation in CBA could influence the electron distribution on the carbonyl carbon and stabilize its zwitterionic form (Testa and Jenner 1988). Thus, studies on the biotransformation of carboxybenzaldehydes present an example of how the inherent properties of chemicals and the variety of catalytic enzymes can give rise to a completely different metabolic profile with structurally similar xenobiotics. Enzyme induction may also modify the metabolic fate of the substances, profoundly. T h e toxicological implications of hydroxymethyl compound formation (Gorrod 1979) deserve further study.
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References AHMED, N. K., FELSTED, R. L., and BACHUR, N. R., 1979, Comparison and characterization of mammalian xenobiotic ketone reductase. Journal of Pharmacology and Experimental Therapeutics, 209, 12-1 9. BRANDEN, C.-L., JORNVALL, H . , EKLUND, H . , and FURUGREN, B., 1975, Alcohol dehydrogenase, in The Enzymes, Vol. 11, edited by P. D. Boyer (New York: Academic Press), pp. 103-190. DAWSON, R. M., ELLIOTT, D. C., ELLIOTT, W. H . , and JONES, K. M . (eds), 1972, Protein estimation by extinction at 260pm and 280pm, in Data for Biochemical Research, Vol. 11 (Oxford: Oxford University Press), pp. 125-126. ENGLANDER, S. W., CALHOUN, D. B., and ENGLANDER, J . J . , 1987, Biochemistry without oxygen. Analytical Biochemistry, 161, 300-306. GORROD, J . W., 1979, Toxic products produced during metabolism of drugs and foreign compounds, in Drug Toxicology, edited by J . W. Gorrod (London: Taylor & Francis), pp. 1-24. GREENFIELD, N., PONTICORVO, L., CHASALOW, F., and LIEBERMAN, S., 1980, Activation and inhibition of the adrenal steroid 21 -hydroxylation system by cytosolic constituents; influence of glutathione, glutathione reductase, and ascorbate. Archives of Biochemistry and Biophysics, 200, 232-244. HIRATA, M., TONDA, K . , and H I C A K J., I , 1986, Induction of a 2-carboxyhenzaldehyde reductase by phenobarbital in primary culture hepatocytes. Biochemical and Biophysical Research Communications, 141, 488493. LOWRY, O., ROSEBROUGH, N. J., FARR,A. L., and RANDALL, R. J., 1951, Protein estimation with the fohn phenol reagent. Journal of Biological Chemistry, 193, 265-275. SHIOBARA, Y., 1977, T h e effect of carboxyl substituent on the metabolism of aromatic aldehydes. Xenobiotica, 7 , 457468. SHIOBARA, Y., and Ocrso, T., 1979, T h e effect of carboxyl substituent on the metabolism in oitro of certain aromatic aldehydes. Xenobiotica, 9, 157-164. TESTA, B., and JENNER, P., 1988, A structural approach to selectivity in drug metabolism and disposition, in Progress in Drug Metabolism, Vol. 11, edited by G . G. Gibson (New York: Taylor & Francis), pp. 75-99. TONDA, K., and HIRATA, M., 1987, Metabolism of phthalidyl theophylline in rat liver. Journal of Pharmacobio-Dynamics, 10, 15-20, TONDA, K., and HIRATA,M . , 1991, Effects of L-methionine on 2-carboxybenzaldehyde reductase induction by phenobarbital in primary cultures of rat hepatocytes. Chemico-Biological Interactions, 77, 149-158. TONDA, K., KAMATA, S., and HIRATA, M., 1987, Metabolism of 1-phthalidyl 5-fluorouracil in rat liver and enzyme induction b y phenobarbital. Xenobiotica, 17, 759-768.
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Purification and molecular properties of 2carboxybenzaldehyde (CBA) reductase from phenobarbital-treated rat liver K. Tonda & M. Hirata To cite this article: K. Tonda & M. Hirata (1992) Purification and molecular properties of 2carboxybenzaldehyde (CBA) reductase from phenobarbital-treated rat liver, Xenobiotica, 22:6, 691-699, DOI: 10.3109/00498259209053131 To link to this article: http://dx.doi.org/10.3109/00498259209053131
Published online: 22 Sep 2008.
Submit your article to this journal
Article views: 2
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ixen20 Download by: [McMaster University]
Date: 06 April 2016, At: 12:56
XENOBIOTICA,
1992, VOL. 22,
NO.
6, 691-699
Purification and molecular properties of 2-carboxybenzaldehyde (CBA) reductase from phenobarbital-treated rat liver K. T O N D A and M. HIRATA" Shionogi Research Laboratories, Shionogi & Co., Ltd, Fukushima-ku, Osaka 553, Japan
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Received 5 November 1991; accepted 20 March 1992
1. A rat liver cytosol enzyme, tentatively named CBA reductase, catalyses the conversion of 2-carboxybenzaldehyde (CBA) to 2-hydroxymethyl benzoic acid in the presence of NADH (or NADPH). CBA reductase is useful for exploring the mechanism of in vitro enzyme induction, as the enzyme can be induced by phenobarbital (PB) both in vivo and in vitro. 2. Possible involvement of glutathione (GSH) in gene expression was suggested by a recent study with cultured rat hepatocytes. 3. CBA reductase was purified about 200-fold by a combination of column chromatography and isoelectric focusing in the presence of mercaptoethanol.
4. The ability to form 2-hydroxymethyl benzoic acid was lost when the enzyme was chromatographed on a hydroxylapatite column in the absence of mercaptoethanol; however, it was restored if sulphydryl compounds or bovine serum albumin was added to the eluate from the column.
5. Gel filtration showed the molecular sizes of CBA reductase from the lO5OOOg supernatant fraction of rat liver extracts and the purified preparation were 64 kDa and 49 kDa, respectively. 6. The results suggest that sulphydryl substances and some proteins play important roles in preserving the molecular and catalytic properties of CBA reductase.
Introduction We have previously shown that a rat liver enzyme in the cytosol catalyses the reduction of 2-carboxybenzaldehyde (CBA) to 2-hydroxymethyl benzoic acid (HMB) in the presence of NADH (Tonda et al. 1987). 3- or 4-Carboxybenzaldehyde could not serve as substrate of the enzyme (Tonda and Hirata 1987), although they are oxidized to their corresponding acids in rat liver (Shiobara and Ogiso 1979). T h e enzyme, tentatively named CBA reductase, was induced by phenobarbital (PB) both in vivo and in vitro (Hirata et al. 1986). CBA reductase activity can be determined either by a spectrophotometric method, which measures the substrate-dependent consumption of NADH, or by h.p.l.c., which quantifies the amount of HMB formed. T h e catalytic activity of the enzyme was abolished when treated with sulphydryl reagents, while 10 mM pyrazole, an alcohol dehydrogenase inhibitor (Branden et al. 1975), or 1 mM barbital, an aldehyde reductase inhibitor (Ahmed et al. 1979), had no effect at all. Since the substrate specificity and responses to various inhibitors differed from those of known dehydrogenases, we attempted to purify the enzyme from liver extracts of PB-treated rat, by a combination of gel filtration, hydrophobic interaction, hydroxylapatite column chromatography and isoelectric focusing. Hydroxylapatite treatment disclosed unique molecular properties of CBA reductase. This report describes the purification of CBA reductase from PB-treated rat liver and various factors regulating the enzyme activity.
* T o whom correspondence should be addressed. 0049%3254/92 $3.00 0 1992 Taylor & Francis Ltd
692
K . Tonda and M . Hirata
Materials and methods Chemicals Phenobarbital-sodium (PB), 2-carboxybenzaldehyde (CBA), hydroxymethylbenzoic acid (HMB), 2mercaptoethanol and hydroxylapatite (100-350 mesh) were purchased from Nacalai Tesque Inc. (Kyoto, Japan), p-Chloromercuribenzoic acid (p-CMB), N-ethylmaleimide, pyridoxal-5-phosphate, NADH and NADPH from Sigma; dithiothreitol and glyoxalic acid from Wako Pure Chem. (Osaka, Japan); 2formylphenoxyacetic acid, Sephadex G-150 superfine and Phenyl Sepharose CL-4B from Pharmacia (Uppsala, Sweden); PIC reagent (Low UVA) was obtained from Waters Assoc. Inc. (Milford, MA) and Toyopearl HW-50 from Toyo Soda Inc. (Tokyo, Japan). Other chemicals were of reagent grade.
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Animals Male Wistar rats, 10-11 weeks old and weighing 300-3309, were used. The rats were treated with PB (80mg/kg per day) in saline injected i.p. for 3 days, and were killed 24h after the last injection. Preparation of liver extracts The rat liver was excised, after perfusion with saline under ether anaesthesia, and homogenized with 5 vol. of 50mM phosphate buffer, pH 7.4, containing 3 mM mercaptoethanol by Potter homogenizer or Ultra-turrax (TKA-Werk). The homogenate was centrifuged first for 2 min at 250g then at 11 OOOg for lOmin at 4°C. The supernatant was further centrifuged at lO5OOOg for 80min at 4°C. Purification of CBA reductase Ammonium sulphate fractionation. Ammonium sulphate was added to the 105 OOOg supernatant at 4°C and the precipitate at 3&50% saturation was collected by centrifugation at 7000g for 10min. T h e precipitate was dissolved in 50mM phosphate buffer with 3 mM mercaptoethanol, pH 7.0, and dialysed overnight at 4°C against the same buffer. Gel chromatography. T h e dialysed enzyme preparation was concentrated by centrifugation with an Amicon Centriflo filter (CF-25) at 450g, and a 6-ml portion was chromatographed on a Toyopearl TSKGel HW 50 column ( 2 6 x 65 cm) with buffer A (50mM phosphate buffer, pH 6.8, containing 3 mM mercaptoethanol). CBA reductase activity in each 5 ml fraction was immediately assayed. Phenyl Sepharose column chromatography. The active fraction from gel chromatography was diluted 5fold with distilled water and applied to a Phenyl Sepharose CL-4B column (2.2 x 15 cm), which had been equilibrated with 5-fold diluted buffer A. After washing the column with buffer A, the enzyme was eluted into lOml tubes with 50% propylene glycol containing 3 mM mercaptoethanol. The eluate was dialysed overnight at 4°C against buffer B (1OmM phosphate buffer-3 mM mercaptoethanol, p H 70). Hydroxylapatite column chromatography. The above enzyme preparation was adsorbed onto a hydroxylapatite column (2.2 x 5.5 cm) which had been equilibrated with buffer B, and washed with the buffer. CBA reductase was then eluted with 38 mM phosphate buffer containing 3 mM mercaptoethanol, pH 6.8. Zsoelectric focusing. The active fraction obtained from hydroxylapatite chromatography was concentrated 5-fold using Amicon Centriflo CF-25, and applied to an LKB isoelectric focusing column (1 10 cm) at 4°C with sucrose density gradient in 0.05% Ampholine (pH 3-10). Electrophoresis was conducted for 20 h at 500 V and 5 mA, then for 2 h at 900 V and 1 mA in the presence of 3 mM mercaptoethanol at 4°C. After fractionation into 1-ml portions, the p H value and U.V.absorbance at 280nm of the fraction was determined. The fraction was dialysed before protein determination. Determination of CBA reductase activity CBA reductase was assayed both by spectrophotometry and h.p.1.c.; the former measures the substrate-dependent consumption of NADH and the latter the formation of HMB from CBA. Spectrophotometry. A portion of the enzyme preparation was preincubated with NADH ( 1 6 0 ~ in ~) 50mM phosphate buffer, pH 7.0, at 37"C, and the reaction was initiated by adding CBA (1 mM, final). The absorbance at 340nm was monitored with a Shimadzu UV 300 spectrophotometer. H.p.1.c. A portion of the enzyme preparation was preincubated with NADH (2 mM) in 50 mM phosphate buffer, pH 7.0, for 5 min at 37"C, then CBA (1 mM) was added. After 10min of reaction at 37"C, a 1-ml portion was transferred to a tube with 0.25 ml 0 4 M perchloric acid containing 125 pg sulphanilamide (internal standard for h.p.1.c.) and 2ml ethyl acetate. The tube was vigorously shaken for lOmin, centrifuged for 5 min at 700g,then a 300 p! portion of the organic layer was evaporated in uucuo, and dissolved in the h.p.1.c. mobile phase (600~1)supplemented with lop1 M-NaOH. H.p.1.c. was conducted on a Cosmosil 5 Ph column (Nacalai Tesque Inc., 4.6 x 1SOmrn), with water-acetonitrile (10: 1 v/v)/Pic reagent-A (low u.v.) (98 : 2 v/v) as the mobile phase and detected at 230 nm by a Shimadzu SCL-6A liquid chromatography system at 25°C. The retention times for CBA, HMB and sulphanilamide were 560,480 and 290 s, respectively, at a flow rate of 1 ml/min. Protein was determined according to Lowry et al. (1951)
693
Purification of carboxybenzaldehyde reductase
using bovine serum albumin as the standard. T h e protein concentration of the eluate from the isoelectric focusing system was assayed by measuring the ratio of absorbance at 260 and 280nm as described by Dawson et al. (1972).
Results Purification of CBA reductase
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CBA reductase was purified about 200-fold from the 105 OOOg supernatant fraction of liver extracts of PB-treated rats, by ammonium sulphate fractionation, Toyopearl-HW 50 gel filtration, Phenyl Sepharose CL-4B hydrophobic chromatography, hydroxylapatite chromatography and isoelectric focusing in the presence of 3 mM mercaptoethanol (table 1). T h e overall yield of enzyme activity was 8% and the isoelectric point (Pi)of the purified enzyme was 6.96 (figure 1). T h e apparent K , value for CBA was 1.7 mM at p H 7.0 with 2 mM NADH and 3 mM mercaptoethanol, Table 1. Purification of 2-carboxybenzaldehyde (CAB) reductase from PB-treated rat liver. Total protein (md
Steps of purification 105 OOOg Supernatant Ammonium sulphate (3@500/,) Toyopearl IW-50 Phenyl Sepharose CL-4B Hydroxylapatite Isoelectric focusing
Total activity' (pmol/min)
5817 2167 1397 257 37 2.Sb
Specific activity Yield (pmol/min per mg) (%) Purification
307 309 251 147 95 26
100 101 82 48 31 8
005 0.14 018 0.57 2.58 1039
1.o 2.7 3.4 10.0 48.9 197.0
"CBA reductase activity was determined in the presence of 3 mM mercaptoethanol (h.p.1.c. method). Protein concentration was calculated by the equation based on the extinction at 260 nm and 280 nm according to Dawson et al. (1972).
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Figure 1. Isoelectric focusing profile of 2-carboxybenzaldehyde (CBA) reductase obtained from hydroxylapatite column chromatography. Fractions (1 ml/tube) were analysed for CBA reductase and protein by measuring the NADH oxidation and U.V.absorbance at 280 nm, respectively.
K . Tonda and M . Hirata
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694
3 4 5 2-Carboxybenzaldehyde [ 1 / S : mM-’] 1
2
Figure 2. Enzyme kinetics of 2-carboxybenzaldehyde (CBA) reductase as a function of (A) CBA ~ ~ and (B)NADH concentration in the presence of concentration in the presence of 1 6 0 NADH 2mM CBA. Enzyme purified from isoelectric focusing was used, and the enzyme activity was assayed with 3 mM mercaptoethanol (h.p.1.c. method). For CBA: K,= 1.68 mM, VmaX =27.6 pnol/min per mg protein. For NADH: K,,,= 0.50 mM, VmaX = 12.7pmol/min per mg protein.
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Hydroxylapatite column chromatography profile of 2-carboxybenzaldehyde (CBA) reductase obtained from Phenyl Sepharose CL-4B hydrophobic chromatography. The enzyme was purified without mercaptoethanol, and the activity was determined in the presence or absence of mercaptoethanol.
Figure 3.
Purification of carboxybenzaldehyde reductase Table 2.
Effect of sulphydryl compounds and proteins on 2-carboxybenzaldehyde (CBA) reductase obtained from hydroxylapatite column chromatography. Experiment 1 Additive
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Experiment 2
Activity (percentage of control)
Additive
Activity (percentage of control)
0
None 3 mM 1 mM 3 mM 3 mM
695
Mercaptoethanol Dithiothreitol CSH (reduced) GSH (oxidized)
3 mM S-Methyl-GSH 3 mM Cysteine 3 mM Cystine 3 mM Methionine
100" 103 103
81 41 100 69 62
Bovin serum albumin (O.Olb) Bovin serum albumin (0.05) Bovin serum albumin (0.10) Bovin serum albumin (0.50) Bovin serum albumin (0.50, boiled for 10 min) Ovalbumin (0.50) y-Globulin (0.50) 3 mM Mercaptoethanol
2 92 105 106 108
0 15 100'
CBA reductase activity was determined in the absence of mercaptoethanol (h.p.1.c.method). 2.39 pmol/min per mg protein. 'mg/rnl in the reaction mixture. '5.31 pmol/min per mg protein. a
while the K,,, value for NADH with 1 mM CBA was 0-5mM (figure 2). In a previous study the K,,, value in the 105 OOOg supernatant fraction for CBA was found to be 1 mM (Hirata et al. 1986).
Substrate specificity T h e catalytic activity of the enzyme appears to be specific for CBA. T h e activity toward 3- or 4-carboxybenzaldehyde was negligibly small, as described previously (Tonda and Hirata 1987). Pyrazole and o-phenanthroline, which inhibit alcohol dehydrogenase, or barbital, an inhibitor of aldehyde reductase, did not inhibit CBA reductase. Variation of N A D H concentration may profoundly influence the enzyme activity as it was increased by 225% (1.6-5.2pmol/min per mg protein) when the concentration of NADH increased from 0.2 to 2 . 0 m ~though , it was increased by only 20% (1.1-1.4pmol/min per mg protein) with increased concentrations of NADPH (0-2-2.0m~).T h e effect of molecular oxygen on CBA reductase was examined using a glucose oxidase-catalase system to deplete oxygen from the reaction mixture (Englander et al. 1987), but the treatment did not affect the enzyme activity at all. Inactivation and reactivation of the enzyme When CBA reductase was purified on a hydroxylapatite column without mercaptoethanol, the enzyme activity capable of producing HMB from CBA was completely lost (0.1 1-+O*OO), though CBA-dependent oxidation of N A D H (0.09-+0*58) took place (figure 3). T h e activity was, however, recovered (0-0.49 pmol HMB/min per ml) when a minute amount of 105 OOOg supernatant was added to the enzyme assay reaction mixture. Treatment of the supernatant with pronase (200 pg/ml) completely abolished the activity, while sulphydryl compounds, G S H , mercaptoethanol, dithiothreitol and cysteine, restored CBA reductase activity (table 2). Although less active, oxidized GSH, methionine and S-methyl G S H also reactivated the enzyme. It is particularly interesting that 0.005% bovine serum
K . Tonda and M . Hirata
696
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i I tubu number
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2 4 6 10 Log. M.W. ( ~ 1 0 4 )
Figure 4. Molecular weight estimation of 2-carboxybenzaldehyde (CBA) reductase obtained from hydroxylapatite column chromatography. The eluate from the column was 5-fold concentrated by centrifugation with Centriflo CF-25 (Amicon Corp') and a 1-ml portion was subjected to chromatography on a Sephadex (3-150 column (1.4 x 45 cm) CBA reductase activity was determined with 3 mM mercaptoethanol. One typical result of three experiments is presented. BSA = bovine serum albumin, or-Chymo = achymotrypsin, Ova = ovalbumin.
albumin reactivated CBA reductase, and furthermore the heat-treated albumin, 10min at 100°C, was still active. Ovalbumin did not reactivate the enzyme. Reactivated CBA reductase produced HMB with consumption of an equimolar amount of NADH. Molecular size of CBA reductase T h e molecular weight of CBA reductase was examined by gel filtration. T h e enzyme fraction eluted from the hydroxylapatite column was concentrated by Centriflo CF-25 (Amicon) and applied on a Sephadex G-150 column (1.4 x 45 cm). T h e CBA reductase activity was determined in the presence of 3 mM mercaptoethanol. T h e molecular weight of purified enzyme was estimated to be 49 kDa (figure 4). However, when the lO5OOOg supernatant of liver extracts was applied to the column, the activity was detected in the fraction corresponded to 64 kDa. This result suggests that CBA reductase could be dissociated to its subunits, or that the shape of the enzyme molecule markedly changed during the chromatography on hydroxylapatite column. Inhibitors of CBA reductase The effects of various aldehydes on purified CAB reductase were examined. They included glyoxylic acid, pyridoxal- 5-phosphate, glyceraldehyde-3-phosphate, oxaloacetic acid, pyruvic acid, dihydroacetone, benzaldehyde, 2-formylphenoxy acetic acid and p-nitrobenzaldehyde. Though they inhibited CBA reductase in the 105 OOOg supernatant, they were ineffective against the purified enzyme except that pyridoxal phosphate, benzaldehyde and formylphenoxy acetic acid inhibited the enzyme in a non-competitive manner. T h e apparent K ivalues for pyridoxal phosphate and formylphenoxy acetic acid were 0.75 and 0.58 mM, respectively
697
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Purification of carboxybenzaldehyde reductase
1
0
2
2-Carboxybenzaldehyde [ 1 / S : mM-’]
(A)
4
2 mM
>
1 2
1 rnM
F
0.75mM 0
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(B) Figure 5.
Effect of (A) 2-formylphenoxy acetic (2-FDA) and (B) pyridoxal-5-phosphate (PALP) on 2-carboxybenzaldehyde (CBA) reductase.
The enzyme preparation obtained from hydroxylapatite column chromatography and isoelectric focusing was used for (A) and (B), respectively. The enzyme activity was determined with 3 m M mercaptoethanol (h.p.1.c. method). V: pmol HMB formed/min per mg protein.
698
K. Tonda and M . Hirata
(figure 5 ) . They did not serve as the substrate for CBA reductase. Divalent metals interfere with the enzyme; C u z + at 1 mM inhibited the enzyme 75% and, Zn2+ and M o Z + about 30%, but EDTA or EGTA (1 mM) did not affect the activity significantly. In the absence of mercaptoethanol the enzyme was completely inhibited by p-CMB and N-ethylmaleimide. Though pyrazole, an alcohol dehydrogenase inhibitor, at 10 mM caused no inhibition at all, 1mM o-phenanthroline, another inhibitor, caused as much as 70% inhibition of the purified CBA reductase.
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Discussion Aromatic aldehydes are generally oxidized to their corresponding acids in animal tissues (Shiobara 1977). Unlike others, 2-carboxybenzaldehyde is reduced to its alcohol, and, despite the enzyme being a cytosolic one, it is inducible by phenobarbital (Hirata et al. 1986). T h e involvement of G S H in the enzyme induction was recently suggested, using primary culture of rat hepatocytes (Tonda and Hirata 1991). T o examine further the mechanism of induction, elucidation of the biochemical properties of CBA reductase would seem to be of value. T h e purified enzyme showed several characteristics.
1. T h e enzyme became physically unstable; for example, though the liver extracts passed through a Sephadex G-150 column and stored at -20°C retained their initial activity for over 2 months, the purified one lost 20% of its activity in 7 days and more than 60% after 14 days. 2. T h e apparent K , value increased from 1 mM to 1 . 7 m by ~ purification. 3. Molecular size of CBA reductase, as judged by gel filtration, in the 105 OOOg supernatant was estimated to be 64 kDa while that of the purified enzyme was 49 kDa. 4. Conversion of CBA to HMB was not observed with the purified enzyme, although the substrate-dependent oxidation of NADH took place. 5 . HMB-forming activity was recovered when the purified enzyme was treated with sulphydryl compounds including G S H , oxidized G S H , S-methyl G S H and methionine. 6 . Reactivation was also attained by bovine serum albumin, though ovalbumin was not effective. Thus, our results indicate that some regulatory subunit, which probably maintains thiol functions, could be dissociated during the enzyme purification. Sulphydryl substances may support the charge transfer from the NADH-reduced enzyme to the substrate CBA. A steroid 21 -hydroxylase purified from bovine adrenal glands showed similar catalytic properties in that the activity was restored by thiol compounds and bovine serum albumin (Greenfield et al. 1980), but subcellular distribution of the enzyme differs from that of CBA reductase. Steroid 21hydroxylase is integrated in microsomes of the tissue and its activity is NADPHdependent, while CBA reductase resides in the cytosol and NADH is preferentially used for its activity. T h e physiological significance of CBA reductase is not clear, but since endogenous substances so far examined could not serve as its substrate, the enzyme may be responsible for the metabolism of xenobiotics. The in vivo metabolism of formylbenzoic acids is particularly interesting in view of the regioselectivity exhibited. As mentioned above, CBA, which bears a carboxylic acid moiety in the ortho position, was exclusively reduced to its alcohol
Purification of carboxybenzaldehyde reductase
699
(HMB) in mammalian tissues and then excreted, while meta- and para-substituted analogues were oxidized to their corresponding dicarboxylic acids. Intramolecular hydrogen bond formation in CBA could influence the electron distribution on the carbonyl carbon and stabilize its zwitterionic form (Testa and Jenner 1988). Thus, studies on the biotransformation of carboxybenzaldehydes present an example of how the inherent properties of chemicals and the variety of catalytic enzymes can give rise to a completely different metabolic profile with structurally similar xenobiotics. Enzyme induction may also modify the metabolic fate of the substances, profoundly. T h e toxicological implications of hydroxymethyl compound formation (Gorrod 1979) deserve further study.
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References AHMED, N. K., FELSTED, R. L., and BACHUR, N. R., 1979, Comparison and characterization of mammalian xenobiotic ketone reductase. Journal of Pharmacology and Experimental Therapeutics, 209, 12-1 9. BRANDEN, C.-L., JORNVALL, H . , EKLUND, H . , and FURUGREN, B., 1975, Alcohol dehydrogenase, in The Enzymes, Vol. 11, edited by P. D. Boyer (New York: Academic Press), pp. 103-190. DAWSON, R. M., ELLIOTT, D. C., ELLIOTT, W. H . , and JONES, K. M . (eds), 1972, Protein estimation by extinction at 260pm and 280pm, in Data for Biochemical Research, Vol. 11 (Oxford: Oxford University Press), pp. 125-126. ENGLANDER, S. W., CALHOUN, D. B., and ENGLANDER, J . J . , 1987, Biochemistry without oxygen. Analytical Biochemistry, 161, 300-306. GORROD, J . W., 1979, Toxic products produced during metabolism of drugs and foreign compounds, in Drug Toxicology, edited by J . W. Gorrod (London: Taylor & Francis), pp. 1-24. GREENFIELD, N., PONTICORVO, L., CHASALOW, F., and LIEBERMAN, S., 1980, Activation and inhibition of the adrenal steroid 21 -hydroxylation system by cytosolic constituents; influence of glutathione, glutathione reductase, and ascorbate. Archives of Biochemistry and Biophysics, 200, 232-244. HIRATA, M., TONDA, K . , and H I C A K J., I , 1986, Induction of a 2-carboxyhenzaldehyde reductase by phenobarbital in primary culture hepatocytes. Biochemical and Biophysical Research Communications, 141, 488493. LOWRY, O., ROSEBROUGH, N. J., FARR,A. L., and RANDALL, R. J., 1951, Protein estimation with the fohn phenol reagent. Journal of Biological Chemistry, 193, 265-275. SHIOBARA, Y., 1977, T h e effect of carboxyl substituent on the metabolism of aromatic aldehydes. Xenobiotica, 7 , 457468. SHIOBARA, Y., and Ocrso, T., 1979, T h e effect of carboxyl substituent on the metabolism in oitro of certain aromatic aldehydes. Xenobiotica, 9, 157-164. TESTA, B., and JENNER, P., 1988, A structural approach to selectivity in drug metabolism and disposition, in Progress in Drug Metabolism, Vol. 11, edited by G . G. Gibson (New York: Taylor & Francis), pp. 75-99. TONDA, K., and HIRATA, M., 1987, Metabolism of phthalidyl theophylline in rat liver. Journal of Pharmacobio-Dynamics, 10, 15-20, TONDA, K., and HIRATA,M . , 1991, Effects of L-methionine on 2-carboxybenzaldehyde reductase induction by phenobarbital in primary cultures of rat hepatocytes. Chemico-Biological Interactions, 77, 149-158. TONDA, K., KAMATA, S., and HIRATA, M., 1987, Metabolism of 1-phthalidyl 5-fluorouracil in rat liver and enzyme induction b y phenobarbital. Xenobiotica, 17, 759-768.
