Purification and properties of two multiple forms of dihydrodiol dehydrogenase from guinea-pig testis

NADP+-dependent dihydrodiol dehydrogenase (trans-1,2-dihydrobenzene-1,2-diol: NADP+ oxidoreductase, EC 1.3.1.20) activity in the cytosol of guinea-pig testis was separated into two major and two minor peaks by Q-Sepharose chromatography; one minor form was immunologically cross-reacted with hepatic aldehyde reductase. The two major enzyme forms were purified to homogeneity. One form, which had the highest amount in the tissue, was a monomeric protein with a molecular weight of 32,000 and isoelectric point of 4.2, showed strict specificity for benzene dihydrodiol and NADP+, and reduced pyridine aldehydes, glyceraldehyde and diacetyl at low rates. Another form, with a molecular weight of 36,000 and isoelectric point of 5.0, oxidized n-butanol, glycerol and sorbitol as well as benzene dihydrodiol in the presence of NADP+ or NAD+, and exhibited much higher reductase activity towards various aldehydes, aldoses and diacetyl. The pI 5.0 form was more sensitive to inhibition by sorbinil and p-chloromercuriphenyl sulfonate than the pI 4.2 form and was activated by sulfate ion. The two enzymes did not catalyze the oxidation of hydroxysteroids and xenobiotic alicyclic alcohols and were immunologically different from hepatic 17 beta-hydroxysteroid-dihydrodiol dehydrogenase. The results indicate that guinea-pig testis contains at least two dihydrodiol dehydrogenases distinct from the hepatic enzymes, one of which, the pI 5.0 enzyme form, may be identical to aldose reductase.     

Purification and properties of multiple forms of dihydrodiol dehydrogenase from human liver

Two acidic and three basic forms of monomeric dihydrodiol dehydrogenase with molecular weights in the range of 36,000-39,000 were purified from human liver. One acidic enzyme (pI 5.2), which was specific for NADP- and dihydrodiols of benzene and naphthalene, was immunologically identified as aldehyde reductase. The other four enzymes oxidized alicyclic alcohols as well as the dihydrodiols using both NADP+ and NAD+ as cofactors, but showed differences in specificity for hydroxysteroids and inhibitor sensitivity. Two of the basic enzymes (pI 9.7 and 9.1) exhibited a 20 alpha-hydroxysteroid dehydrogenase activity and sensitivity to 1,10-phenanthroline, whereas the third basic enzyme (pI 7.6) oxidized some 3 alpha-hydroxysteroids at low rates and was inhibited by cyclopentane-1,1-diacetic acid. Another acidic enzyme, which accounted for the largest amount of enzyme activity in the tissue and appeared in two heterogenous forms with pI values of 5.9 and 5.4, showed a high 3 alpha-hydroxysteroid dehydrogenase activity and was the most sensitive to inhibition by medroxyprogesterone acetate. The Km values of the enzymes, except the pI 5.2 enzyme, for hydroxysteroids (10(-6) to 10(-7) M) were lower than those for xenobiotic alcohols.     

Isolation of multiple forms of indanol dehydrogenase associated with 17 beta-hydroxysteroid dehydrogenase activity from male rabbit liver

Seven multiforms of indanol dehydrogenase were isolated in a highly purified state from male rabbit liver cytosol. The enzymes were monomeric proteins with similar molecular weights of 30,000-37,000 but with distinct electrophoretic mobilities. All the enzymes oxidized alicyclic alcohols including benzene dihydrodiol and hydroxysteroids at different optimal pH, but showed clear differences in cofactor specificity, steroid specificity, and reversibility of the reaction. Two NADP+-dependent enzymes exhibited both 17 beta-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes and 3 alpha-hydroxysteroid dehydrogenase activity for 5 beta-androstan-3 alpha-ol-17-one. Three of the other enzymes with dual cofactor specificity catalyzed predominantly 5 beta-androstane-3 alpha,17 beta-diol dehydrogenation. The reverse reaction rates of these five enzymes were low, whereas the other two enzymes, which had 3 alpha-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes or 3(17)beta-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes, highly reduced 3-ketosteroids and nonsteroidal aromatic carbonyl compounds with NADPH as a cofactor. All the enzymes exhibited Km values lower for the hydroxysteroids than for the alicyclic alcohols. The results of kinetic analyses with a mixture of 1-indanol and hydroxysteroids, pH and heat stability, and inhibitor sensitivity suggested strongly that, in the seven enzymes, both alicyclic alcohol dehydrogenase and hydroxysteroid dehydrogenase activities reside on a single enzyme protein. On the basis of these data, we suggest that indanol dehydrogenase exists in multiple forms in rabbit liver cytosol and may function in in vivo androgen metabolism.     

Separation and properties of multiple forms of dihydrodiol dehydrogenase from hamster liver

1. Five multiple forms of dihydrodiol dehydrogenase (EC 1.3.1.20) with similar molecular weights of around 35,000 were purified from hamster liver cytosol. 2. All the enzymes oxidized trans-dihydrodiols of benzene and naphthalene and reduced various carbonyl compounds, but showed clear differences in specificities for other alcohols and cofactors, and in inhibitor sensitivity. 3. Two NADP+-dependent enzymes were immunologically identified with aldehyde reductase (EC 1.1.1.2) and 3 alpha-hydroxytsteroid dehydrogenase (EC 1.1.1.50). 4. The other enzymes with dual cofactor specificity oxidized xenobiotic alicyclic alcohols, and one of them was active on 3 alpha- and 17 beta-hydroxysteroids with NAD+ as a preferable cofactor.     

Molecular cloning of a novel type of rat cytoplasmic 17beta-hydroxysteroid dehydrogenase distinct from the type 5 isozyme

Rat liver contains two cytosolic enzymes (TBER1 and TBER2) that reduce 6-tert-butyl-2,3-epoxy-5-cyclohexene-1,4-dione into its 4R- and 4S-hydroxy metabolites. In this study, we cloned the cDNA for TBER1 and examined endogenous substrates using the homogenous recombinant enzyme. The cDNA encoded a protein composed of 323 amino acids belonging to the aldo-keto reductase family. The recombinant TBER1 efficiently oxidized 17beta-hydroxysteroids and xenobiotic alicyclic alcohols using NAD+ as the preferred coenzyme at pH 7.4, and showed low activity towards 20alpha- and 3alpha-hydroxysteroids, and 9-hydroxyprostaglandins. The enzyme was potently inhibited by diethylstilbestrol, hexestrol and zearalenone. The coenzyme specificity, broad substrate specificity and inhibitor sensitivity of the enzyme differed from those of rat NADPH-dependent 17beta-hydroxysteroid dehydrogenase type 5, which was cloned from the liver and characterized using the recombinant enzyme. The mRNA for TBER1 was highly expressed in rat liver, gastrointestinal tract and ovary, in contrast to specific expression of 17beta-hydroxysteroid dehydrogenase type 5 mRNA in the liver and kidney. Thus, TBER1 represents a novel type of 17beta-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution. In addition, TBER2 was identified as 3alpha-hydroxysteroid dehydrogenase on chromatographic analysis of the enzyme activities in rat liver cytosol and characterization of the recombinant 3alpha-hydroxysteroid dehydrogenase.     

Identity of dihydrodiol dehydrogenase and 3 alpha-hydroxysteroid dehydrogenase in rat but not in rabbit liver cytosol

Dihydrodiol dehydrogenase and 3 alpha-hydroxysteroid dehydrogenase activity in rat and rabbit liver cytosol have been analyzed by isoelectric focussing and subsequent activity staining. Identity of the two enzymes in rat liver cytosol is demonstrated. At least 4 main enzyme forms possessing dihydrodiol dehydrogenase activity can be detected in rabbit liver cytosol. However, in this species, only one of these forms has measurable activity towards 3 alpha-hydroxysteroids.     

Dihydrodiol dehydrogenase activities of rabbit liver are associated with hydroxysteroid dehydrogenases and aldo-keto reductases.

1. Dihydrodiol dehydrogenase activities were investigated in rabbit liver. Using a five-step purification scheme, eight isoenzymes of dihydrodiol dehydrogenase with isoelectric points of 5.55-9.3 and promoter molecular masses of 34-35 kDa were purified to apparent homogeneity and designated CF-1 to CF-6, CM-1 and CM-2. 2. CF-1 and CF-2 had near-neutral isoelectric points of 7.4 and 6.8 and molecular masses of about 125 kDa in the native state. Both enzymes readily accepted NAD+ as well as NADP+ as coenzymes, had relatively low Km values of 0.33 mM and 0.47 mM for benzene dihydrodiol and resembled previously described carbonyl reductases in their substrate specificity towards ketones and quinones. 3. CF-5 and CF-6 had acidic isoelectric points of 5.9 and 5.55 and native molecular masses of approximately 60 kDa. They displayed a strong preference for NADP(H) as coenzyme and had high Km and Vmax with benzene dihydrodiol. Since these enzymes reduced p-nitrobenzaldehyde and glucuronic acid efficiently, they appeared to be closely related to aldehyde reductase. 4. CF-4 had a high 3 alpha-hydroxysteroid dehydrogenase activity for the diagnostic substrate androsterone, a moderate activity for other 3 alpha-hydroxysteroids as well as 17 alpha-hydroxysteroids, and relatively low activities for 3 beta-hydroxysteroids and 17 beta-hydroxysteroids. CF-5 and CM-1 had high 17 beta-hydroxysteroid dehydrogenase activity for the diagnostic substrate 5 alpha-dihydrotestosterone, and low to moderate activities for other 17 beta-hydroxysteroids as well as 3 alpha-hydroxysteroids. 5. The isoenzyme CM-2 had an isoelectric point of 9.3 and was a very active quinone reductase with phenanthrene-9,10-quinone as substrate. It was potently inhibited by phenobarbital. 6. We conclude that the dihydrodiol dehydrogenase activities of rabbit liver are associated with aldehyde and carbonyl reductase and with 3 alpha-hydroxysteroid and 17 beta-hydroxysteroid dehydrogenases.     

Demonstration of 3 alpha(17 beta)-hydroxysteroid dehydrogenase distinct from 3 alpha-hydroxysteroid dehydrogenase in hamster liver.

NAD(+)-linked and NADP(+)-linked 3 alpha-hydroxysteroid dehydrogenases were purified to homogeneity from hamster liver cytosol. The two monomeric enzymes, although having similar molecular masses of 38,000, differed from each other in pI values, activation energy and heat stability. The two proteins also gave different fragmentation patterns by gel electrophoresis after digestion with protease. The NADP(+)-linked enzyme catalysed the oxidoreduction of various 3 alpha-hydroxysteroids, whereas the NAD(+)-linked enzyme oxidized the 3 alpha-hydroxy group of pregnanes and some bile acids, and the 17 beta-hydroxy group of testosterone and androstanes. The thermal stabilities of the 3 alpha- and 17 beta-hydroxysteroid dehydrogenase activities of the NAD(+)-linked enzyme were identical, and the two enzyme activities were inhibited by mixing 17 beta- and 3 alpha-hydroxysteroid substrates, respectively. Medroxyprogesterone acetate, hexoestrol and 3 beta-hydroxysteroids competitively inhibited 3 alpha- and 17 beta-hydroxysteroid dehydrogenase activities of the enzyme. These results show that hamster liver contains a 3 alpha(17 beta)-hydroxysteroid dehydrogenase structurally and functionally distinct from 3 alpha-hydroxysteroid dehydrogenase.     

Age-dependent changes in the multiple forms of the soluble 17 beta-hydroxysteroid dehydrogenase of female rabbit liver.

The soluble NADP-dependent 17 beta-hydroxysteroid dehydrogenase activity of female rabbit liver increases with the age of the animal, the specific activity of the enzyme in the 56-day-old rabbit being 3 times that of the 28-day-old animal. The increase in activity is accompanied by a change in the molecular heterogeneity of the enzyme. Three forms (enzymes I, II and III) were identified in the liver cytosol of the 56-day-old female rabbit, whereas only one major form (enzyme IIIY) was present in the 28-day-old animal. Peptide maps of the four purified enzymes showed that there were minor differences in structure. The enzyme present in the liver of the 28-day-old rabbit was distinct from the three enzymes of the 56-day-old animal. All of the enzymes exhibited bifunctional activity, having 17 beta-hydroxysteroid dehydrogenase activity towards androgen and oestrogen substrates and 3 alpha-hydroxysteroid dehydrogenase activity towards androgens of the 5 beta-androstane series. The differences in substrate specificity of the enzymes paralleled their differences in structure. The data suggest that one enzyme (enzyme III) may have a special role in steroid metabolism during development in the female rabbit.     

A novel dihydrodiol dehydrogenase in bovine liver cytosol: purification and characterization of multiple forms of dihydrodiol dehydrogenase.

Three enzymes (DD1, DD2, and DD3) having dihydrodiol dehydrogenase activity were purified to homogeneity from bovine cytosol. DD1 and DD2 were identified as 3 alpha-hydroxysteroid dehydrogenase and high-Km aldehyde reductase, respectively, as judged from their molecular weights, substrate specificities and inhibitor sensitivities. DD3 was a unique enzyme which could specifically catalyze the dehydrogenation of trans-benzenedihydrodiol and trans-naphthalenedihydrodiol without any activity toward the other tested alcohols, aldehydes, ketones, and quinones. The Km value of DD3 (0.18 mM) for benzenedihydrodiol was lower than those of other dihydrodiol dehydrogenases so far reported. DD3 immunologically crossreacted with DD1, but showed no crossreactivity with DD2. Additionally, DD3 was inhibited in a competitive manner, with a low Ki value of 1 microM, by androsterone, which was a good substrate for DD1. It was assumed that DD3 is a novel enzyme which is specific to dihydrodiols, exhibiting similarity to DD1 in immunological and structural properties.     

Kinetic and stereochemical characterization of hamster liver 3 alpha-hydroxysteroid dehydrogenase and 3 alpha(17 beta)-hydroxysteroid dehydrogenase

The kinetic mechanism of NADP(+)-dependent 3 alpha-hydroxysteroid dehydrogenase and NAD(+)-dependent 3 alpha(17 beta)-hydroxysteroid dehydrogenase, purified from hamster liver cytosol, was studied in both directions. For 3 alpha-hydroxysteroid dehydrogenase, the initial velocity and product inhibition studies indicated that the enzyme reaction sequence is ordered with NADP+ binding to the free enzyme and NADPH being the last product to be released. Inhibition patterns by Cibacron blue and hexestrol, and binding studies of coenzyme and substrate are also consistent with an ordered bi bi mechanism. For 3 alpha(17 beta)-hydroxysteroid dehydrogenase, the steady-state kinetic measurements and substrate binding studies suggest a random binding pattern of the substrates and an ordered release of product; NADH is released last. However, the two enzymes transferred the pro-R-hydrogen atom of NAD(P)H to the carbonyl substrate.     

Electrophoretic and immunochemical characterization of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenases of rat tissues.

The properties of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase from Sprague-Dawley rat liver cytosol have been re-examined in light of several reports which suggest that multiple forms of the enzyme may exist in this tissue. During enzyme purification, chromatography on DE-52 cellulose and chromatofocusing columns indicated the existence of only one form of the protein. Re-chromatography of the purified enzyme by either of these techniques failed to resolve the protein into additional forms. When the purified enzyme was subjected to SDS/polyacrylamide-gel electrophoresis a single band corresponding to Mr 34,000 was detected. Two-dimensional gels showed one predominant protein with a pI of 5.9. Using the homogeneous enzyme as antigen, high-titre polyclonal antibody was raised in rabbits. Western-blot analysis of cytosolic proteins prepared from male and female Sprague-Dawley rat liver indicated the presence of a single immunoreactive band with an Mr of 34,000 in both sexes. All of the 3 alpha-hydroxysteroid dehydrogenase activity present in rat liver cytosol could be immunotitrated with the antibody and the resulting titration curve was superimposable on the titration curve obtained with the purified enzyme. Western-blot analysis of cytosolic proteins prepared from livers of male Wistar and Fischer rats also revealed the presence of a single immunoreactive protein with an Mr of 34,000. These data indicate that, contrary to previous reports, only one form of the dehydrogenase may exist in liver cytosols prepared from a variety of rat strains. Although 3 alpha-hydroxysteroid dehydrogenase activity is known to be widely distributed in male Sprague-Dawley rat tissues, Western blots indicate that only the liver, lung, testis and small intestine contain immunoreactive protein with an Mr of 34,000. The levels of immunoreactive protein in these tissues follow the distribution of dihydrodiol dehydrogenase.     

Effect of allylisopropylacetamide and Sedormid on enzymes of steroid metabolism in rat liver

Female rats, treated with allylisopropylacetamide (AIA) showed a marked decrease of hepatic NADH-5 alpha-reductase, NADPH-5 alpha-reductase, NAD+- and NADP+-3 alpha-hydroxysteroid dehydrogenase activities and an increase of the activity of NADH- and NADPH-5 beta-reductase and NAD+ and NADP+-3 beta-hydroxysteroid dehydrogenase. Administration of Sedormid decreased the activities of 5 alpha-reductases and 3 alpha-hydroxysteroid dehydrogenases (substrate, 5 alpha-dihydrotestosterone) and increased the activity of NADH-5 beta-reductase, whereas no effect was seen on NADPH-5 beta-reductase and 3 beta-hydroxysteroid dehydrogenase.     

Non-K-region o-quinones as enzyme-generated inactivators of dihydrodiol dehydrogenase

Homogeneous 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase from rat liver cytosol catalyzes the NAD(P)+-dependent oxidation of non-K-region trans-dihydrodiols of polycyclic aromatic hydrocarbons, many of which are proximate carcinogens. These reactions proceed with Km values in the millimolar range to yield highly reactive o-quinones that can be trapped as thioether adducts [Smithgall, T. E., Harvey, R. G., & Penning, T. M. (1988) J. Biol. Chem. 263, 1814-1820]. The enzymatically generated o-quinones, e.g., naphthalene-1,2-dione and benzo[a]pyrene-7,8-dione are potent inhibitors of the dehydrogenase, yielding IC50 values of 5.0 and 10.0 microM, respectively. Naphthalene-1,2-dione was found to be an efficient irreversible inhibitor of the enzyme and can inactivate equimolar concentrations of the dehydrogenase, yielding a t 1/2 for the enzyme of 10 s or less. By contrast (+/-)-trans-1,2-dihydroxy-1,2-dihydronaphthalene promotes a slower inactivation of the dehydrogenase, yielding a Kd of 70 microM and a limiting rate constant that corresponds to a t 1/2 at saturation of 23.2 min. Inactivation by this dihydrodiol has an obligatory requirement for NADP+. Examination of the kcat for the oxidation of (+/-)-trans-1,2-dihydroxy-1,2-dihydronaphthalene yields a partition ratio for the dihydrodiol of 200,000, suggesting that alkylation from the parent dihydrodiol is a rare occurrence. Benzo[a]pyrene-7,8-dione, which is the product of the enzymatic oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, also promotes a time- and concentration-dependent inactivation of the dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of flutamide on 5 alpha-reductases, 5 beta-reductases, and 3-hydroxysteroid dehydrogenases in rat liver

Flutamide (0.5 mM) decreased in vitro the activity of NADH-5 alpha-reductase (substrate testosterone) in liver homogenate of male and female rats, whereas no change of activity of NADPH-5 alpha-reductase was observed. NADH- and NADPH-5 beta-reductase activity increased only in liver of female, but not of male rats. NAD+-3 beta-hydroxysteroid dehydrogenase and NAD+-3 alpha-hydroxysteroid dehydrogenase (substrate 5 alpha-dihydro-testosterone) in liver homogenate from female rats were inhibited by flutamide (0.5 mM), whereas the activity of NADP+-3 alpha-hydroxysteroid dehydrogenase (substrate 5 alpha-dihydrotestosterone) and of NAD+-3 alpha-hydroxysteroid dehydrogenase (substrate 5 beta-dihydrotestosterone) increased in presence of flutamide. The activity of NADH- and NADPH-5 alpha-reductase decreased after flutamide administration to female rats at a dose of 5 mg per day for 7 days.     

Occurrence of hydroxysteroid oxidoreductases in liver of turtles

1. Hydroxysteroid oxidoreductases have been partially purified from the cytosol fraction (105,000 g supernatant) of liver from a fresh-water turtle (Podocnemis expansa) and a sea-water turtle (Chelonia mydas mydas) by precipitation with ammonium sulphate (AS, 10-80% saturation). 2. The following enzymes were detected (substrates in brackets): 3 alpha-hydroxysteroid oxidoreductase (androsterone), 3 beta-hydroxysteroid oxidoreductase (DHEA) and 17 beta-hydroxysteroid oxidoreductase (testosterone, oestradiol-17 beta). NAD as well as NADP were effective as cofactors. 3. In fresh-water turtle, highest activities of the 3 alpha-enzyme were measured in the 20% AS fraction (cofactor NAD), of the 3 beta-enzyme in the 60% AS fraction (cofactor NAD) and of the 17 beta-enzyme in the 40% AS fraction (cofactor NADP). 4. In sea-water turtle, highest activities were observed for all three enzymes in the 60% AS fraction. 5. Generally, enzyme activities were higher in sea-water turtles than in fresh-water turtles. The most active enzyme in both turtles was found to be the 3 alpha-hydroxysteroid oxidoreductase, followed by the 17 beta- and the 3 beta-hydroxysteroid oxidoreductases.     

Purification and characterization of NADP+-dependent 3 alpha-hydroxysteroid dehydrogenase from mouse liver cytosol

A monomeric 3 alpha-hydroxysteroid dehydrogenase with a molecular weight of 34,000 was purified to apparent homogeneity from mouse liver cytosol. The enzyme catalyzed the reversible oxidation of the 3 alpha-hydroxy group of C19-, C21-, and C24-steroids, reduced a variety of carbonyl compounds, and was inhibited by SH-reagents, synthetic estrogens, anti-inflammatory drugs, prostaglandins, and delta 4-3-ketosteroids. Although these properties are similar to those of the enzyme from rat liver cytosol, the mouse enzyme exhibited low dehydrogenase activity toward benzene dihydrodiol and some alicyclic alcohols, it showed a strict cofactor specificity for NADP(H), and high substrate inhibition was observed in the reverse reaction. In addition, dexamethasone, deoxycorticosterone, and medroxyprogesterone acetate inhibited the mouse enzyme competitively at low concentrations and noncompetitively at high concentrations, whereas hexestrol, indomethacin, and prostaglandin A1 were competitive inhibitors. Steady-state kinetic measurements in both directions indicated that the reaction proceeds through an ordered bi bi mechanism with the cofactors binding to the free enzyme. The 3-ketosteroid substrates inhibited the enzyme uncompetitively at elevated concentrations, suggesting that the substrates bind to the enzyme.NADPH complex and to the enzyme NADP+ complex.     

Molecular characterization of the cynomolgus monkey Macaca fascicularis steroidogenic enzymes belonging to the aldo-keto reductase family

Steroidogenic enzymes belonging to the aldo-keto reductase family (AKR) possess highly homologous sequences while having different activities. To gain further knowledge about the function as well as the regulation of these enzymes in the monkey, we have isolated cDNA sequences encoding monkey type 5 17beta-hydroxysteroid dehydrogenase, 20alpha-hydroxysteroid dehydrogenase and 3alpha-hydroxysteroid dehydrogenase, and characterized their enzymatic activity and mRNA tissue distribution. Sequence analysis indicates that these enzymes share approximately 94 and 76% amino acid identity with human and mouse homologs, respectively. Monkey type 5 17beta-HSD possesses 95.9% amino acid sequence identity with human type 5 17beta-HSD. It catalyzes the transformation of 4-androstenedione into testosterone, but it lacks 20alpha-hydroxysteroid dehydrogenase activity that is present in the human enzyme. This activity seems to be specific to human, since mouse type 5 17beta-HSD does not show significant 20alpha-HSD activity. In addition, monkey and mouse 20alpha-HSD possess relatively high 20alpha-, 3alpha-, and 17beta-HSD activities, while their human counterpart is confined to 20alpha-HSD activity. The monkey 3alpha-HSD possesses relatively high 3alpha-, 17beta-, and 20alpha-HSD activities; human type 1 3alpha-HSD exerts 3alpha- and 20alpha-HSD activities; the mouse 3alpha-HSD displays a unique 3alpha-HSD activity. Quantification of mRNA expression shows that the monkey 3alpha-HSD is exclusively expressed in the liver, while the type 5 17beta-HSD is predominately found in the kidney, with lower levels observed in the stomach, liver, and colon. Monkey 20alpha-HSD mRNA is highly expressed in the kidney, stomach, and liver. Our study provides the basis for future investigations on the regulation and function of these enzymes in the monkey.     

Inhibition of microsomal NAD(P)H oxidation by Triton X-100

The non-ionic detergent Triton X-100 is shown to inhibit the spontaneous oxidation of NAD(P)H associated with rat liver microsomes. Advantage of this observation is taken to measure different microsomal NAD(P)H-dependent oxidoreductase activities such as 3-alpha-hydroxysteroid dehydrogenase, dihydrodiol dehydrogenase and various xenobiotic oxidoreductases. This inhibition provides an easy method for the screening of the under-investigated microsomal oxidoreductive metabolism of xenobiotics.     

Evidence that enzyme-generated aromatic Michael acceptors covalently modify the nucleotide-binding site of 3 alpha-hydroxysteroid dehydrogenase.

The non-steroidal allylic and acetylenic alcohols 1-(4'-nitrophenyl)prop-2-en-1-ol (I) and 1-(4'-nitrophenyl)prop-2-yn-1-ol (II) are oxidized by homogeneous 3 alpha-hydroxysteroid dehydrogenase to the corresponding alpha beta-unsaturated ketones 1-(4'-nitrophenyl)prop-2-en-1-one (III) and 1-(4'-nitrophenyl)prop-2-yn-1-one (IV), which then inactivate the enzyme selectively with high affinity; low effective partition ratios are observed for the parent alcohols [Ricigliano & Penning (1989) Biochem. J. 262, 139-149]. Inactivation of 3 alpha-hydroxysteroid dehydrogenase by compound (I) displays an NAD+ concentration optimum. Scavenging experiments indicate that the enzyme-generated inactivators (III) and (IV) alkylate the enzyme via a release-and-return mechanism. Several lines of evidence suggest that compounds (III) and (IV) covalently modify the NAD(P)(+)-binding site. First, micromolar concentrations of NAD(P)H offer substantial protection against enzyme inactivation mediated by Michael acceptors (III) and (IV). In these protection studies Kd measurements for NAD(P)H approached those measured by fluorescence titration of free enzyme. Secondly, under initial-velocity conditions compounds (III) and (IV) act essentially as competitive inhibitors of NAD+ binding, and as mixed competitive or non-competitive inhibitors against androsterone binding. Thirdly, enzyme inactivated with either compound (III) or compound (IV) fails to bind to NAD+ affinity columns (e.g. Affi-gel Blue). Under the same conditions of chromatography native enzyme and enzyme affinity-labelled at the steroid-binding site with 17 beta-bromoacetoxy-5 alpha-dihydrotestosterone is retained on the affinity column. A kinetic scheme that represents the inactivation of the homogeneous dehydrogenase by the enzyme-generated alkylators (III) and (IV) is presented.