Publications

• The enzymatic reduction of delta 4-3-ketosteroids.

  TOMKINS G.M.

  J Biol Chem 225:13-24(1957) [PubMed] [EuropePMC]

• Purification and characterization of delta 4-3-ketosteroid 5 beta-reductase.

  Okuda A., Okuda K.

  delta 4-3-Ketosteroid 5 beta-reductase was purified about 230-fold from 100,000 X g supernatant of rat liver homogenate using 7 alpha-hydroxy-4-cholesten-3-one as substrate throughout. The purified enzyme was electrophoretically homogeneous, and its molecular weight determined by sodium dodecyl su ... >> More

  delta 4-3-Ketosteroid 5 beta-reductase was purified about 230-fold from 100,000 X g supernatant of rat liver homogenate using 7 alpha-hydroxy-4-cholesten-3-one as substrate throughout. The purified enzyme was electrophoretically homogeneous, and its molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 37,000 and that determined by gel filtration chromatography on calibrated Sephadex G-100 column was 37,200. The absorption spectrum of the purified enzyme showed only a peak at 276 nm due to aromatic amino acids, precluding the presence of a prosthetic group such as flavine in the molecule. The enzyme is highly labile in a low buffer concentration, but is markedly stabilized in the presence of 20% glycerol in 10 mM phosphate buffer. Higher buffer concentration such as 300 mM potassium phosphate buffer was also effective to prevent deterioration in the absence of glycerol, but the effect was somewhat lower compared to glycerol. The purified enzyme showed the activity toward a variety of substrates including testosterone, cortisol, cortisone, progesterone, 4-androstene-3,17-dione, 7 alpha-hydroxy-4-cholesten-3-one, and 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one. The optimal pH for the 5 beta-reduction of 7 alpha-hydroxy-4-cholesten-3-one was 7.4, and the cofactor required for the reaction was NADPH, while NADH revealed no effect. The enzyme activity was inhibited by p-chloromercuribenzoate, but its inhibition was prevented by the presence of a reduced form of glutathione. << Less

  J Biol Chem 259:7519-7524(1984) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Identification of cortisone 5 beta-reductase as delta 4-3-ketosteroid 5 beta-reductase.

  Furuebisu M., Deguchi S., Okuda K.

  Cortisone 5 beta-reductase (4,5 beta-dihydrocortisone:NADP+ delta 4-oxidoreductase, EC 1.3.1.3) was purified from rat liver 100,000 X g supernate to a homogeneous state based on the catalytic activity. In the course of purification the activity was always accompanied by androstenedione 5 beta-redu ... >> More

  Cortisone 5 beta-reductase (4,5 beta-dihydrocortisone:NADP+ delta 4-oxidoreductase, EC 1.3.1.3) was purified from rat liver 100,000 X g supernate to a homogeneous state based on the catalytic activity. In the course of purification the activity was always accompanied by androstenedione 5 beta-reductase (3-oxo-5 beta-steroid:NADP+ delta 4-oxidoreductase, EC 1.3.1.23) and no fraction which revealed only cortisone 5 beta-reductase activity but lacked androstenedione 5 beta-reductase was observed. Partial denaturation of the purified enzyme with p-chloromercuribenzoate or wtih heat reduced both enzyme activities to a similar extent. When both substrates were added together at concentrations sufficient to saturate or nearly saturate the enzyme when added separately, the total rate of the reactions was much less than the sum of the rates of the reactions measured separately. Judging from these results it was concluded that cortisone 5 beta-reduction and that of androstenedione are catalyzed by the same catalytic site of a single protein. << Less

  Biochim Biophys Acta 912:110-114(1987) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Genomic organization of a human 5beta-reductase and its pseudogene and substrate selectivity of the expressed enzyme.

  Charbonneau A., The V.L.

  The enzyme 5beta-reductase catalyzes the reduction of the 4-ene of 3-ketosteroids, converting them into 5beta-dihydro-3-ketosteroids and, thus, could be involved in the metabolism of 4-cholestene-3-one, progesterone, 17~-hydroxyprogesterone, aldosterone, corticosterone, cortisol, 4-androstenedione ... >> More

  The enzyme 5beta-reductase catalyzes the reduction of the 4-ene of 3-ketosteroids, converting them into 5beta-dihydro-3-ketosteroids and, thus, could be involved in the metabolism of 4-cholestene-3-one, progesterone, 17~-hydroxyprogesterone, aldosterone, corticosterone, cortisol, 4-androstenedione, and testosterone. In this study, we report the genomic structure of a human 5beta-reductase gene, its tissue distribution, the characterization of an intronless pseudogene and the substrate selectivity of the enzyme. The gene coding for the active 5beta-reductase contains nine exons like most members of the aldo-keto reductase family, but the sequence covered by the gene, more than 42 kb, is much longer than the sequence of other members of this family. There are many large introns, especially introns 3, 4 and 7 that span approx. 7 kb, and intron 1 that contains more than 10 kb. Northern blot analysis showed three band sizes of 1.3, 2.2 and 2.7 kb. The 1.3 and 2.7 kb bands are highly expressed in the liver while weaker 2.2 and 1.3 kb bands have been observed in the testis and colon, respectively. We also identified an intronless gene having 86% homology with the 5beta-reductase cDNA sequence. Since its sequence contains many stop codons, this gene is most probably a pseudogene. To determine more precisely the substrate selectivity of the enzyme, we established a stable cell line expressing human 5beta-reductase in transformed embryonic kidney (HEK-293) cells. The transfected cells efficiently catalyze the transformation of progesterone, androstenedione, 17alpha-hydroxyprogesterone and testosterone. However, they catalyze much less efficiently the transformation of compounds containing an 11beta-hydroxy group, such as aldosterone, corticosterone and cortisol. In addition to its role in cholesterol catabolism, it is well recognized that 5beta-reductase inactivates active androgens. Indeed, 5beta-dihydrotestosterone (5beta-DHT), the product of the reduction of testosterone by 5beta-reductase, is not active while its 5~-isomer (DHT) is the most potent natural androgen. Recent findings show that 5beta-pregnanes are active ligands in the induction of CYP3A through the orphan receptor hPAR. Our results thus open an opportunity for studying the new role of 5beta-reductase in the formation of a new type of active steroids. << Less

  Biochim. Biophys. Acta 1517:228-235(2001) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Separation of delta 4-5 alpha-hydrogenases from rat liver homogenates.

  DORFMAN R.I., FORCHIELLI E.

  J Biol Chem 223:443-448(1956) [PubMed] [EuropePMC]

• The delta4-hydrogenases of guinea pig adrenal gland.

  BROWN-GRANT K., FORCHIELLI E., DORFMAN R.I.

  J Biol Chem 235:1317-1320(1960) [PubMed] [EuropePMC]

• Purification of 5 beta-reductase from hepatic cytosol fraction of chicken.

  Sugimoto Y., Yoshida M., Tamaoki B.

  From the cytosol fraction (supernatant fluid at 105,000 g) of chicken liver, 4-en-3-oxosteroid 5 beta-reductase (EC 1.3.1.23) was purified by ammonium sulfate precipitation, followed by Butyl Toyopearl, DEAE-Sepharose, Sephadex G-75 and hydroxylapatite column chromatographies. The enzyme activity ... >> More

  From the cytosol fraction (supernatant fluid at 105,000 g) of chicken liver, 4-en-3-oxosteroid 5 beta-reductase (EC 1.3.1.23) was purified by ammonium sulfate precipitation, followed by Butyl Toyopearl, DEAE-Sepharose, Sephadex G-75 and hydroxylapatite column chromatographies. The enzyme activity was quantitated from amount of the 5 beta-reduced metabolites derived from [4-14C]testosterone. During the purification procedures, 17 beta-hydroxysteroid dehydrogenase which was present in the cytosol fraction was separated from 5 beta-reductase fraction by the Butyl Toyopearl column chromatography. By the DEAE-Sepharose column chromatography, 3 alpha- and 3 beta-hydroxysteroid dehydrogenases were able to be removed from 5 beta-reductase fraction. The final enzyme preparation was apparently homogeneous on SDS-polyacrylamide gel electrophoresis. Purification was about 13,600-fold from the hepatic cytosol. The molecular weight of this enzyme was estimated as 37,000 Da by SDS-polyacrylamide gel electrophoresis and also by Sephadex G-75 gel filtration. For 5 beta-reduction of 4-en-3-oxosteroids, such as testosterone, androstenedione and progesterone, NADPH was specifically required as cofactor. Km of 5 beta-reductase for NADPH was estimated as 4.22 x 10(-6) M and for testosterone, 4.60 x 10(-6) M. The optimum pH of this enzyme ranged from pH 5.0 to 6.5 and other enzymic properties of the 5 beta-reductase were examined. << Less

  J Steroid Biochem Mol Biol 37:717-724(1990) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Cloning and expression of cDNA of human delta 4-3-oxosteroid 5 beta-reductase and substrate specificity of the expressed enzyme.

  Kondo K.-H., Kai M.-H., Setoguchi Y., Eggertsen G., Sjoeblom P., Setoguchi T., Okuda K., Bjoerkhem I.

  The enzyme delta 4-3-oxosteroid 5 beta-reductase (3-oxo-5 beta-steroid: NADP+ oxidoreductase and 4,5 beta-dihydrocortisone: NADP+ delta 4-oxidoreductase) catalyzes the reduction of the delta 4 double bond of bile acid intermediates and steroid hormones carrying the delta 4-3-one structure in the A ... >> More

  The enzyme delta 4-3-oxosteroid 5 beta-reductase (3-oxo-5 beta-steroid: NADP+ oxidoreductase and 4,5 beta-dihydrocortisone: NADP+ delta 4-oxidoreductase) catalyzes the reduction of the delta 4 double bond of bile acid intermediates and steroid hormones carrying the delta 4-3-one structure in the A/B cis configuration. Human delta 4-3-oxosteroid 5 beta-reductase cDNA was isolated from a liver cDNA library by cross hybridization with a previously cloned rat cDNA, which was used as a probe [Onishi, Y. Noshiro, M., Shimosato, T. & Okuda, K.-I. (1991) FEBS Lett. 283, 215-218]. DNA sequence analysis of a hybridization-positive clone predicted the human delta 4-3-oxosteroid 5 beta-reductase to contain 326 amino acids. The amino acid sequence of the human delta 4-3-oxosteroid 5 beta-reductase had 79% overall identity to the rat enzyme sequence. It also showed 54% and 50% overall identity with rat 3 alpha-hydroxysteroid dehydrogenase and human aldose reductase, respectively. RNA blotting analysis demonstrated the existence of a single delta 4-3-oxosteroid 5 beta-reductase mRNA of approximately 2.7 kb in human liver. Transfection of the cDNA into COS cells resulted in the expression of an active enzyme with a high activity toward the bile acid intermediates 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one and 7 alpha-hydroxy-4-cholesten-3-one. In addition, the expressed enzyme showed a small but significant 5 beta-reduction activity toward 11 beta,17 alpha,21-trihydroxy-delta 4-pregnene-3,20-dione (cortisol) and 17 beta-hydroxy-delta 4-androsten-3-one (testosterone) whereas no activity was observed toward delta 4-pregnene-3,20-dione (progesterone) or delta 4-androstene-3-17-dione (androstenedione). The substrate specificity of the human enzyme is considerably narrower than that of the rat enzyme, and the enzyme seems to be more important for bile acid biosynthesis than for metabolism of steroid hormones. << Less

  Eur. J. Biochem. 219:357-363(1994) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.