Carbonyl Reductase Activity Research Articles

11β-Hydroxysteroid dehydrogenase type 1: tissue-specific expression and reductive metabolism of some anti-insect agent azole analogues of metyrapone

The azole analogues of metyrapone are novel candidates for selective anti-insect agents that inhibit the synthesis of 20-hydroxyecdysone (20E), the moulting hormone of insects. Metyrapone, which is a model substrate for studying the reductive properties of oxidoreductases, is itself effectively reduced to the corresponding alcohol by the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD 1). For this reason, the ability of 11β-HSD 1 to metabolize the metyrapone analogues as well was studied. In addition, the expression (by Western blots) and activity (reduction/oxidation of dehydrocorticosterone/corticosterone) of 11β-HSD 1 in different male and female mouse tissues were investigated. Xenobiotic carbonyl reductase activities in these tissues were assessed with metyrapone as a model substrate. The kinetic parameters of 11β-HSD 1 with metyrapone analogues as substrates were calculated after high-pressure liquid chromatography (HPLC) determination of the product alcohols. Our results indicate that the novel insecticides are extensively metabolized by mouse 11β-HSD 1. Moreover, the resulting alcohols are not only less toxic than the parent ketones but also have the potential, owing to the newly formed hydroxyl group, to be eliminated from the body by consecutive phase II reactions. Thus, the new metyrapone analogues may be potential anti-insect agents, safer for humans due to their reductive detoxification, mainly by the hepatic 11β-HSD 1, and selectively affecting insect development by inhibiting ecdysone 20-monooxygenase (E-20-M).

Teleost ovarian carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase: potential role in the production of maturation-inducing hormone during final oocyte maturation.

17alpha,20beta-Dihydroxy-4-pregnen-3-one is the major oocyte maturation-inducing hormone of several teleost species. Gonadotropin-induced increase in ovarian 20beta-hydroxysteroid dehydrogenase activity is essential for the synthesis of maturation-inducing hormone. Cloning and expression studies suggest that ayu (Plecoglossus altivelis) ovarian carbonyl reductase can function as 20beta-hydroxysteroid dehydrogenase. The amino acid sequence deduced from the isolated cDNA had 276 amino acid residues and shared approximately 60% homology with mammalian and teleostean carbonyl reductases. The sequence data search showed that the ayu cDNA clone belongs to the short-chain dehydrogenase/reductase family. The clear lysate prepared from Escherichia coli harboring the cDNA catalyzed the production of maturation-inducing hormone. Its identification was confirmed by two-dimensional, thin-layer chromatography followed by recrystallization. Purification of the E. coli-expressed cDNA product revealed that it possessed both carbonyl reductase and steroid dehydrogenase activities, and 17alpha-hydroxyprogesterone, the endogenous immediate precursor of maturation-inducing hormone, was one of the preferred substrates. Furthermore, Northern blot analysis denoted that the transcripts are present both in fully grown, immature ovarian follicles and at higher levels in mature ovarian follicles. These results demonstrate that the carbonyl reductase of ayu ovary is involved in the production of maturation-inducing hormone, and they provide evidence for a novel physiological role of this enzyme in the final maturation of oocytes. Based on its functional properties, the enzyme can be referred to as carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase.

Reductive metabolism of an alpha,beta-ketoalkyne, 4-phenyl-3-butyn-2-one, by rat liver preparations.

The reduction of the triple bond and carbonyl group of an alpha,beta-ketoalkyne, 4-phenyl-3-butyn-2-one (PBYO), by rat liver microsomes and cytosol was investigated. The triple-bond-reduced product trans-4-phenyl-3-buten-2-one (PBO) and the carbonyl-reduced product 4-phenyl-3-butyn-2-ol (PBYOL) were formed when PBYO was incubated with rat liver microsomes in the presence of NADPH. The triple bond of 1-phenyl-1-butyne, deprenyl, ethynylestradiol, ethinamate, and PBYOL, in which the triple bond is not adjacent to a carbonyl group, were not reduced by liver microsomes even in the presence of NADPH. PBO was further reduced to 4-phenyl-2-butanone (PBA) by liver cytosol with NADPH. PBYOL was also formed from PBYO by liver cytosol in the presence of NADPH or NADH. The microsomal triple-bond reductase activity was inhibited by disulfiram, 7-dehydrocholesterol, and 18 beta-glycyrrhetinic acid but not beta-diethylaminoethyldiphenylpropylacetate or carbon monoxide. The triple-bond reductase activity in liver microsomes was not enhanced by several inducers of the rat cytochrome P450 system. These results suggested that the triple-bond reduction is caused by a new type of reductase, not cytochrome P450. The microsomal and cytosolic carbonyl reductase activities were not inhibited by quercitrin, indomethacin, or phenobarbital. Only S-PBYOL was formed from PBYO by liver cytosol. In contrast, liver microsomes produced R-PBYOL together with the S-enantiomer to some extent. Ethoxyresorufin-O-dealkylase activity in rat liver microsomes was markedly inhibited by PBYO and PBO, partly by PBYOL, but not by PBA.

Differential hormonal regulation of carbonyl reductase activities in liver and kidney microsomes of rat

1. In the male rat, hepatic microsomal carbonyl reductase (CR) activity decreased by testectomy (Tx) was restored to the control level by the treatment with testosterone propionate (TP), even though the enzyme activity decreased by hypophysectomy (Hx) was not increased by the treatment with TP. On the other hand, renal microsomal CR activities decreased by Tx and Hx were markedly increased by the treatment with TP. 2. The treatment with TP had no effect on the CR activity in liver microsomes of the ovariectomized or hypophysectomized female rat. On the other hand, the CR activities in kidney microsomes of the ovariectomized and hypophysectomized female rat were significantly increased by the treatment with TP. 3. The results indicate that in rat programmed by neonatal androgens, the hepatic microsomal CR activity is regulated indirectly by androgens through the hypothalamus-pituitary system, whereas the hormonal regulation of the renal microsomal CR activity is not via the pituitary. We conclude that the regulatory mechanism of the CR activity in liver microsomes is distinguishable from that in kidney microsomes.

Sperm-zona pellucida interaction involves a carbonyl reductase activity in the hamster.

For successful fertilization to occur, the spermatozoa must transit through an egg-specific extracellular matrix or zona pellucida (zp) to reach and ultimately fuse with the oocyte plasma membrane. This process involves ligand-receptor recognition between the zp and the acrosomal cap of the sperm. The hamster sperm protein P26h, a receptor which is acquired during epididymal transit, has been suggested to act in sperm-zp binding. The cloning and characterization of the full-length cDNA-encoding hamster P26h revealed 85% identity with a porcine lung carbonyl reductase. To better understand the mechanism by which P26h interacts with zp proteins, we investigated carbonyl reductase activity during gamete interactions. In the present study, we show that specific inhibitors of carbonyl reductase such as diclofenac and phenylbutazone decreases sperm-zp binding without affecting the motility, progressivity or acrosome integrity of sperm. We also detected, and partly purified, carbonyl reductase activities from cauda epididymal sperm protein extract and this activity was associated with an enriched fraction of P26h. Removing P26h from the partly purified protein fractions by immunoaffinity chromatography led to the loss of carbonyl reductase activity. The findings that sperm-zp binding is blocked by carbonyl reductase inhibitors and that P26h is active in mature sperm suggest that P26h could play an important role in the fertilization process.

Characterization of multiple Chinese hamster carbonyl reductases

Carbonyl reductase (CR) is an enzyme which can catalyze the oxidoreduction of various carbonyl compounds in the presence of NAD(P)H. With the PCR method, using primers carrying the conserved nucleotide sequence among mammalian CRs, we isolated three different cDNAs (CHCR1, CHCR2 and CHCR3) which encode a unique carbonyl reductase from the Chinese hamster. The PCR products of CHCR1 and CHCR2 were clearly isolated with Bpu1102I, BspEI and XmaI restriction enzymes. The nucleotide-sequence of CHCR3 was completely different from those of CHCR1 and CHCR2. The predicted double-wound βαβα-structures of the CHCRs suggests the presence of a typical NADP +-binding motif and is similar to the corresponding region of 3α,20β-hydroxysteroid dehydrogenase and mouse lung tetrameric carbonyl reductase. The deduced amino acid sequence of CHCR1 showed a high homology to CHCR2 (>96%) and the other mammalian CRs (>81%). However, CHCR3 showed a high homology to human CBR3 (>86%) and a relatively lower homology to the other CHCRs (<76%). Bacterial recombinant CHCRs showed typical carbonyl reductase activities towards 4-benzoylpyridine, 4-nitrobenzaldehyde and pyridine 4-carboxyaldehyde. These three CRs showed not only 3-keto reductase of steroids, but also 20-keto reductase. However, these CRs did not show any activity of 17-keto reductase activity. Both CHCR1 and CHCR2 have prostaglandin 9-keto reductase and 15-hydroxyprostaglandin dehydrogenase activities towards PGE 2 and PGF 2α from the analyses of enzymatic reaction products. The results of Western blotting and RT-PCR suggest these CHCRs have a tissue-dependent-distribution in the Chinese hamster.

Mutation of threonine-241 to proline eliminates autocatalytic modification of human carbonyl reductase

Carbonyl reductase catalyses the reduction of steroids, prostaglandins and a variety of xenobiotics. An unusual property of human and rat carbonyl reductases is that they undergo modification at lysine-239 by an autocatalytic process involving 2-oxocarboxylic acids, such as pyruvate and 2-oxoglutarate. Comparison of human carbonyl reductase with the pig enzyme, which does not undergo autocatalytic modification, identified three sites, alanine-236, threonine-241 and glutamic acid-246, on human carbonyl reductase that could be important in the reaction of lysine-239 with 2-oxocarboxylic acids. Mutagenesis experiments show that replacement of threonine-241 with proline (T241P) in human carbonyl reductase eliminates the formation of carboxyethyl-lysine-239. In contrast, the T241A mutant has autocatalytic activity similar to wild-type carbonyl reductase. The T241P mutant retains catalytic activity towards menadione, although with one-fifth the catalytic efficiency of wild-type carbonyl reductase. Replacement of threonine-241 with proline is likely to disrupt the local structure near lysine-239. We propose that integrity of this local environment is essential for chemical modification of lysine-239, but not absolutely required for carbonyl reductase activity.

Mutation of threonine-241 to proline eliminates autocatalytic modification of human carbonyl reductase

Carbonyl reductase catalyses the reduction of steroids, prostaglandins and a variety of xenobiotics. An unusual property of human and rat carbonyl reductases is that they undergo modification at lysine-239 by an autocatalytic process involving 2-oxocarboxylic acids, such as pyruvate and 2-oxoglutarate. Comparison of human carbonyl reductase with the pig enzyme, which does not undergo autocatalytic modification, identified three sites, alanine-236, threonine-241 and glutamic acid-246, on human carbonyl reductase that could be important in the reaction of lysine-239 with 2-oxocarboxylic acids. Mutagenesis experiments show that replacement of threonine-241 with proline (T241P) in human carbonyl reductase eliminates the formation of carboxyethyl-lysine-239. In contrast, the T241A mutant has autocatalytic activity similar to wild-type carbonyl reductase. The T241P mutant retains catalytic activity towards menadione, although with one-fifth the catalytic efficiency of wild-type carbonyl reductase. Replacement of threonine-241 with proline is likely to disrupt the local structure near lysine-239. We propose that integrity of this local environment is essential for chemical modification of lysine-239, but not absolutely required for carbonyl reductase activity.

Heterogeneity of 11β-hydroxysteroid dehydrogenase type 1/microsomal carbonyl reductase (11β-HSD/CR) in guinea pig tissues. Purification of the liver form suggests modification in the cosubstrate binding site

11β-hydroxysteroid dehydrogenase (11β-HSD) and xenobiotic carbonyl reductase activities were determined in guinea pig tissue microsomes. The data indicate the presence of a NADP(H) dependent form, distinct from the known type I isozyme. Purification of 11β-HSD-1 from liver microsomes resulted in two distinct peaks, resolved by dye–ligand chromatography, indicating differences in the cosubstrate binding site. Immunoblot analysis using anti 11β-HSD-1 antibodies reveals the presence of similar structural determinants between the enzyme forms. Both have an apparent molecular mass of 32 kDa, suggesting protein modifications occurring in the type 1 isozyme which account for the differences in chromatographic behaviour.

Purification and characterization of oxidoreductases-catalyzing carbonyl reduction of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in human liver cytosol

1. Four enzymes were purified to homogeneity from human liver cytosol and were demonstrated to be responsible for carbonyl reduction of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK). 2. Carbonyl reductase (EC 1.1.1.184), a member of the short-chain dehydrogenase reductase (SDR) superfamily, was compared with three isoenzymes of the aldo-keto reductase (AKR) superfamily in terms of enzyme kinetics, co-substrate dependence and inhibition pattern. 3. AKR1C1, 1C2 and 1C4, previously designated as dihydrodiol dehydrogenases (DD1, DD2 and DD4), showed lower Km (0.2, 0.3 and 0.8 mm respectively) than did carbonyl reductase (7 mM), whereas carbonyl reductase exhibited the highest enzyme efficiency (Vmax/Km) for NNK. Multiplication of enzyme efficiencies with the relative quantities of individual enzymes in cytosol resulted in a rough estimate of their contributions to total alcohol metabolite formation. These were ~ 60% for carbonyl reductase, 20% each for AKR1C1 and 1C2, and 1% for AKR1C4. 4. Except for AKR1C4, the enzymes had a strong preference for NADPH over NADH, and the highest activities were measured with an NADPH-regenerating system. Carbonyl reductase activity was extensively inhibited by menadione, rutin and quercitrin, whereas medroxyprogesterone acetate, phenolphthalein and flufenamic acid were potent inhibitors of AKR1C1, 1C2 and 1C4. 5. In conclusion, cytosolic members of the SDR and AKR superfamilies contribute to reductive NNK detoxification in human liver, the enzymes responsible being carbonyl reductase and aldoketo reductases of the AKR1C subfamily.

A carbonyl reductase-catalyzing reduction of N3-phenacyluridine in rabbit liver.

Carbonyl reductase activity for a novel hypnotic, N3-phenacyluridine, was mainly localized in the cytosol fraction of rabbit liver. The enzyme (N3-phenacyluridine reductase) which catalyzes the reduction of N3-phenacyluridine to N3-alpha-hydroxy-beta-phenethyluridine was purified from the cytosolic fraction of rabbit liver by various chromatographic techniques (DEAE-Sephacel, Red Sepharose CL-6B and hydroxylapatite). N3-Phenacyluridine reductase had a minimum molecular weight of 39kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. This enzyme required reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor and its optimal pH was 7.5. Flavonoids (quercetin and quercitrin) were potent inhibitors of the enzyme, but pyrazole or barbital had little effect. The apparent Km and Vmax values of the enzyme for the reduction of N3-phenacyluridine were 0.32 mM and 8.7 units/mg protein, respectively. A variety of carbonyl compounds, including N3-phenacyluridine, were effectively reduced by the enzyme. However, the enzyme purified from rabbit liver differs in several respects from known carbonyl reductases in rabbit liver.

Microsomal carbonyl reductase responsible for reduction of 4-phenyl-3-buten-2-one in rats.

trans-4-Phenyl-3-buten-2-one (PBO), a flavoring additive, was transformed to the carbonyl-reduced product, trans-4-phenyl-3-buten-2-ol (PBOL) by rat liver microsomes, but not by liver cytosol, in the presence of NADH or NADPH. PBOL formed was identified by comparison with an authentic sample. The reductase activity was not inhibited by quercitrin, an inhibitor of cytosolic carbonyl reductase. The carbonyl reduction product of PBO by liver microsomes was identified as the R-enantiomer of PBOL by HPLC analysis. Rat blood also exhibited the carbonyl reductase activity in the presence of NADH or NADPH, but to a lesser extent.

Cloning and sequencing of the cDNA species for mammalian dimeric dihydrodiol dehydrogenases

Cynomolgus and Japanese monkey kidneys, dog and pig livers and rabbit lens contain dimeric dihydrodiol dehydrogenase (EC 1.3.1.20) associated with high carbonyl reductase activity. Here we have isolated cDNA species for the dimeric enzymes by reverse transcriptase-PCR from human intestine in addition to the above five animal tissues. The amino acid sequences deduced from the monkey, pig and dog cDNA species perfectly matched the partial sequences of peptides digested from the respective enzymes of these animal tissues, and active recombinant proteins were expressed in a bacterial system from the monkey and human cDNA species. Northern blot analysis revealed the existence of a single 1.3 kb mRNA species for the enzyme in these animal tissues. The human enzyme shared 94%, 85%, 84% and 82% amino acid identity with the enzymes of the two monkey strains (their sequences were identical), the dog, the pig and the rabbit respectively. The sequences of the primate enzymes consisted of 335 amino acid residues and lacked one amino acid compared with the other animal enzymes. In contrast with previous reports that other types of dihydrodiol dehydrogenase, carbonyl reductases and enzymes with either activity belong to the aldo-keto reductase family or the short-chain dehydrogenase/reductase family, dimeric dihydrodiol dehydrogenase showed no sequence similarity with the members of the two protein families. The dimeric enzyme aligned with low degrees of identity (14-25%) with several prokaryotic proteins, in which 47 residues are strictly or highly conserved. Thus dimeric dihydrodiol dehydrogenase has a primary structure distinct from the previously known mammalian enzymes and is suggested to constitute a novel protein family with the prokaryotic proteins.

Interindividual variability in the expression and NNK carbonyl reductase activity of 11β-hydroxysteroid dehydrogenase 1 in human lung

The balance between metabolic activation and detoxification is critical in determining the susceptibility to lung cancer upon exposure to the tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Carbonyl reduction of NNK, followed by glucuronidation, is the main detoxification pathway of this lung carcinogen in humans. Recently, we have identified 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD 1) as microsomal NNK carbonyl reductase in liver and lung. In the present study, the interindividual variability of 11β-HSD 1 expression and NNK-carbonyl reductase activity was examined in human lung by RT–PCR, Western blot analysis and enzyme activity. Levels of 11β-HSD 1 mRNA varied over an almost 20-fold range among different subjects. Levels of NNK carbonyl reductase activity in lung microsomes closely resembled the relative amounts of immunoreactive protein as determined by Western blot analysis. In view of the large interindividual differences in the susceptibility of tobacco smoke related lung cancer, we present the first data on the variability of 11β-HSD 1 expression and NNK carbonyl reduction in human lung.

Cloning and sequencing of the cDNA species for mammalian dimeric dihydrodiol dehydrogenases

Cynomolgus and Japanese monkey kidneys, dog and pig livers and rabbit lens contain dimeric dihydrodiol dehydrogenase (EC 1.3.1.20) associated with high carbonyl reductase activity. Here we have isolated cDNA species for the dimeric enzymes by reverse transcriptase-PCR from human intestine in addition to the above five animal tissues. The amino acid sequences deduced from the monkey, pig and dog cDNA species perfectly matched the partial sequences of peptides digested from the respective enzymes of these animal tissues, and active recombinant proteins were expressed in a bacterial system from the monkey and human cDNA species. Northern blot analysis revealed the existence of a single 1.3 kb mRNA species for the enzyme in these animal tissues. The human enzyme shared 94%, 85%, 84% and 82% amino acid identity with the enzymes of the two monkey strains (their sequences were identical), the dog, the pig and the rabbit respectively. The sequences of the primate enzymes consisted of 335 amino acid residues and lacked one amino acid compared with the other animal enzymes. In contrast with previous reports that other types of dihydrodiol dehydrogenase, carbonyl reductases and enzymes with either activity belong to the aldo-keto reductase family or the short-chain dehydrogenase/reductase family, dimeric dihydrodiol dehydrogenase showed no sequence similarity with the members of the two protein families. The dimeric enzyme aligned with low degrees of identity (14-25%) with several prokaryotic proteins, in which 47 residues are strictly or highly conserved. Thus dimeric dihydrodiol dehydrogenase has a primary structure distinct from the previously known mammalian enzymes and is suggested to constitute a novel protein family with the prokaryotic proteins.

Carbonyl reductase and NADPH cytochrome P450 reductase activities in human tumoral versus normal tissues

The use of bioreductive agents in enzyme-directed bioreductive therapy has been proposed to take advantage not only of hypoxia in tumours, but also of the presence of reductases that metabolise such compounds. In this study, we studied the activities of NADPH cytochrome P450 reductase (P450R) and carbonyl reductase (CR) in 17 human lung tumours and 18 human breast tumours, together with the corresponding normal tissues. For lung cancer but not for breast cancer there was a significant difference in the CR activity between normal and tumour tissue. CR activity was increased with respect to the normal tissue between 2-fold and 40-fold indicating heterogeneity in tumour samples. No relationship was found between CR activity and the histological type, tumoral grade or TNM stage of the tumours. Although some variation in P450R activity in tumoral versus normal tissues was found in the majority of the samples studied, no significant differences could be demonstrated.

Characterization of S-hexylglutathione-binding proteins of human hepatocellular carcinoma: separation of enoyl-CoA isomerase from an Alpha class glutathione transferase form.

Recent studies have revealed binding of mitochondrial enoyl-CoA isomerase (ECI) to S-hexylglutathione-Sepharose, an affinity matrix used for purification of glutathione transferases (GSTs), and the enzyme has been suggested to be identical with the Alpha class form of GST with a subunit molecular mass of about 30 kDa. In the present study, S-hexylglutathione-binding proteins of human hepatocellular carcinomas were characterized to examine their identity. Supernatant fractions of carcinoma and surrounding tissues were applied to an affinity column, and bound fractions were resolved into three proteins with subunit molecular masses/pI values of 33 kDa/7.0, 30 kDa/5.8 and 29 kDa/5.8 in addition to the well-characterized four GST subunits, A1, A2, M1 and P1, by two-dimensional gel electrophoresis. The proteins were further purified by chromatofocusing at pH 7.4-4.0. The 30 and 29 kDa proteins were eluted at pH 4.9 and by 1 M NaCl respectively, and could be clearly separated from each other. The 29 kDa protein exhibited a low but significant activity towards 1-chloro-2,4-dinitrobenzene (4.25 micromol/min per mg of protein) and reacted with anti-(GST A1-2) antibody, suggesting that it is a member of the GST Alpha class. The 30 kDa protein did not react with anti-GST antibodies and was identified as ECI by immunoblotting and N-terminal-amino-acid-sequencing analyses. The results thus indicated that the Alpha class GST form composed of the 29 kDa subunits and ECI are two different proteins. The 33 kDa protein was eluted from the chromatofocusing column at pH 7.0 and did not react with either anti-GST antibodies or antibodies against mitochondrial enzymes involved in the beta-oxidation of fatty acids. However, it exhibited a carbonyl reductase activity with menadione and ubiquinone, and amino acid sequences of its peptides cleaved by Staphylococcus aureus V8 proteinase were consistent with those reported for the enzyme. Thus this protein binding to S-hexylglutathione-Sepharose was identified as carbonyl reductase.

Heterologous expression of carbonyl reductase: Demonstration of prostaglandin 9-ketoreductase activity and paraquat resistance

Transfection of murine NIH3T3 fibroblasts with a pSV2-derived eukaryotic expression vector for human cytosolic carbonyl reductase (E.C. 1.1.1.141) resulted in clones with increased carbonyl reductase activity as demonstrated by an elevation in cellular NADPH-dependent alcohol (menadione) reductase activity. Prostaglandin 9-ketoreductase (9KR) activity, previously noted only in purified enzyme preparations, was also elevated. Although the cellular molar capacity of 9KR activity was less than menadione reductase activity (picomoles versus nanomoles per mg of protein), when compared to endogenous activity there was a greater relative increase in 9KR activity as compared to menadione activity (10 fold increase versus 3 fold). Thus, the 9KR properties of carbonyl reductase may have a physiologic role in prostaglandin regulation. Most transgenic clones lost their enhanced carbonyl reductase activity despite continuous selection, but two clones retained enhanced enzyme activity. RNA analysis indicated that these two murine clones expressed human carbonyl reductase mRNA. These two clones overexpressing carbonyl reductase did not display resistance to menadione, in agreement with a previous report. There was, however, a demonstrable increase in resistance to paraquat of a magnitude similar to that previously noted with transgenic cell lines overexpressing manganese Superoxide dismutase.

Carbonyl reductase activity exhibited by pig testicular 20 beta-hydroxysteroid dehydrogenase.

The carbonyl reductase activity exhibited by pig testicular 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) was examined using a recombinant enzyme. Kinetic parameters were obtained for 48 carbonyl group-containing substrates, including aromatic aldehydes, aromatic ketones, cycloketones, quinones, aliphatic aldehydes and aliphatic ketones. 20 beta-HSD showed a high affinity towards quinones, such as 9,10-phenanthrenequinone, alpha-naphthoquinone and menadione (Km values of 4, 2 and 5 microM, respectively), and the substrate utilization efficiency (Vmax/Km) of the enzyme against these quinones was very high. Cyclohexanone and 2-methylcyclohexanone were also reduced with a high Vmax/Km value, but not cyclopentanone or 2-methylcyclopentanone. Various aromatic aldehydes and ketones including benzaldehyde- and acetophenone-derivatives were reduced by 20 beta-HSD. Especially, 4-nitrobenzaldehyde and 4-nitroacetophenone were reduced with high Vmax/Km values in the related compounds. The enzyme also reduced the pyridine-derivatives, 2-, 3-, and 4-benzoylpyridine, with the Vmax/Km value for 2-benzoylpyridine being the highest. 20 beta-HSD reduced aliphatic aldehydes and aliphatic ketones, but was more effective on the former. The correlation between the structure of carbonyl compounds and their substrate Vmax/Km is discussed.

Ovarian carbonyl reductase activity in rats at persistent oestrus.

Age-related changes and effects of gonadotrophins on ovarian carbonyl reductase (CR) of virgin female Wistar-Imamichi rats were investigated. In mature rats ovarian CR is an LH-dependent oxidoreductase and is closely involved in the periovulatory process. After 8 months of age, all of the rats exhibited persistent oestrus and the ovarian mass was significantly decreased, whereas 4- and 5-month-old rats showed the regular 4 day oestrous cycles and ovulation occurred. The ovarian CR activity towards four substrates declined steeply consistent with persistent oestrus and the enzyme activity for each substrate at 15 months of age was 1.4-53.7% lower than at 4 months of age. The ovarian CR concentration measured by western blot analysis was also lower at 8 months of age. Equine chorionic gonadotrophin (eCG) and hCG were administered to 10- and 15-month-old rats at persistent oestrus to examine whether the ovarian CR in rats at persistent oestrus is influenced by gonadotrophins. Treatment with hCG not only increased ovarian CR activity and its concentration but also caused ovulation at both ages. The immunoreactivity to anti-ovarian CR antibody in the theca cells of the hCG-treated ovary was greater than that of the control ovary. Equine(e) CG did not stimulate ovarian CR activity in rats at persistent oestrus, although ovarian mass of 10-month-old rats was increased after administration of 50 iu of eCG. These results indicate that changes in ovarian CR with ageing are related to the transition of regular oestrous cyclicity to acyclicity and that the expression of ovarian CR markedly decreased in aged rats is stimulated by hCG.