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.     

The influence of oestradiol on androgen- and oestrogen-dependent enzyme activities of hepatic steroid metabolism in rats.

Five sexually differentiated enzyme activities of hepatic steroid metabolism (cytoplasmic 17 beta-hydroxysteroid dehydrogenase, 5 beta-reductase; microsomal 3 alpha- and 3 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase) were investigated in intact, gonadectomized and hypophysectomized rats after administration of a single dose of oestradiol valerate. Oestradiol administration caused a partial or complete feminization of these activities in intact male rats. The influence of oestradiol on these activities in gonadectomized rats was determined by the mode of sex hormone-dependent regulation of the individual activity: the most prominent effects were seen in the oestrogen-dependent activities (17 beta-hydroxysteroid dehydrogenase, 5 beta-reductase); no effect was seen in the completely androgen-dependent 3 alpha-hydroxysteroid dehydrogenase because gonadectomy alone was sufficient to cause complete feminization of the activity. Oestradiol administration had no effect on the activities of hypophysectomized rats. The fact that oestrogen administration to intact male rats caused greater changes than prepuberal gonadectomy demonstrates that oestrogen action is more than simple suppression of testicular function.     

Time-studies of the changes in the sex-dependent activities of enzymes of hepatic steroid metabolism in the rat during oestrogen administration.

This investigation was undertaken to elucidate the amount of oestradiol and duration of its administration necessary to cause complete feminization of the activities of cytoplasmic 3 alpha- and 17 beta-hydroxysteroid dehydrogenase, microsomal 3 alpha- and 3 beta-hydroxysteroid dehydrogenase and microsomal 5 alpha-reductase in male rat liver. With the exception of cytoplasmic 3 alpha-hydroxysteroid dehydrogenase, 5 microgram oestradiol/d for 8 days and less was sufficient to cause complete feminization. The order of oestrogen sensitivity was cytoplasmic 3 alpha-hydroxysteroid dehydrogenase greater than microsomal 3 beta-hydroxysteroid dehydrogenase greater than microsomal 3 alpha-hydroxysteroid dehydrogenase greater than microsomal 5 alpha-reductase greater than cytoplasmic 17 beta-hydroxysteroid dehydrogenase. Although the changes occurring after oestradiol administration are qualitatively the same as after testectomy, they occur more rapidly. This rules out the possibility that oestradiol exerts its effect via androgen deprivation. Diethylstilboestrol administration causes the same changes in cytoplasmic 17 beta- and microsomal 3 beta-hydroxysteroid dehydrogenase activity as oestradiol, although the dosage must be increased 100 fold. The effect of diethylstilboestrol on 5 alpha-reductase activity changes with the dose applied. Doses up to 100 microgram/d partially feminize the activity, but at higher doses the enzyme activity is repressed.     

The hypophysis in the regulation of androgen and oestrogen dependent enzyme activities of steroid hormone metabolism in rat liver cytosol.

In order to determine whether the gonadal and hypophyseal modes of regulation recently reported for the microsomal enzymes of hepatic steroid metabolism are also valid for cytoplasmic enzymes, three enzymes whose activities exhibit sex differences (male:female activity ratio shown in brackets), 5beta-reductase(1.7:1), 20alpha-hydroxysteroid dehydrogenase(5 : 1) and 17beta-hydroxysteroid dehydrogenase (4:1), as well as one enzyme whose activity shows no sex difference, 3beta-hydroxy-delta5-steroid dehydrogenase, were investigated after various interferences with the endocrine balance (gonadectomy, hypophysectomy, combination of both operations, administration of testosterone or oestradiol). From the results of this and a previous study the following statements can be made about the endocrine control of hepatic enzyme activities. Those enzymes whose activities show sex differences are either androgen or oestrogen dependent; the sex hormone acts in either an inductive or repressive manner. 1) Criteria for androgen dependency are the feminization of enzyme activity after testectomy or inhibition of testicular function by administration of oestradiol; masculinization of the enzyme activity after administration of testosterone to male or female castrates. Using these criteria the following enzymes investigated in this laboratory fall into this category: all microsomal enzymes which show sex differences in their activity (3alpha-, 3beta-, delta4-3beta, 20-hydroxysteroid dehydrogenase; cortisone alpha-reductase; steroid hydroxylases and 16alpha-hydroxylase) as well as the cytoplasmic 20alpha-hydroxysteroid dehydrogenase. Apart from the single exception of 20alpha-hydroxy-steroid dehydrogenase the presence of the hypophysis is obligatory for the androgen to be effective. The hypophysis does not only work in a permissive manner, but participates in establishing the sex specific activity levels in a manner which is antagonistic to the androgen action. 2) Criteria for oestrogen dependency are that the female animal reacts to gonadectomy, as well as to the inhibition of ovarian function after testosterone administration, by a masculinization of the enzyme activities. After administration of oestradiol, but not gonadectomy, the male animal exhibits typical female activity. Using these criteria the cytoplasmic 5beta-reductase and 17beta-hydroxysteroid dehydrogenase are oestrogen dependent. The repressive oestrogen effect observed on 17beta-hydroxysteroid dehydrogenase is antagonistic to hypophyseal action, whereas in the case of 5beta-reductase it is synergistic. 3) The activities of cytoplasmic 3beta-hydroxy-delta5-steroid dehydrogenase and microsomal 7alpha-hydroxylase show no sex differences and are not influenced by any interference with the endocrine balance.     

Inhibition of pyridine-nucleotide-dependent enzymes by pyrazoles. Synthesis and enzymology of a novel A-ring pyrazole steroid

A novel A-ring pyrazole steroid, 2,3-bisaza-A-nor-1,5(10)-estradien-17 beta-ol (3), was synthesized as a potential inhibitor of steroidal NAD(P)H-dependent oxidoreductases. Compound 3 proved to be a potent inhibitor of 3(17)beta-hydroxysteroid dehydrogenase (from P. testosteroni) exhibiting a Ki of 90 +/- 20 nM. The activities of 3 alpha,20 beta-hydroxysteroid dehydrogenase (from S. hydrogenans), steroid-5 alpha-reductase (from rat prostate), and 3 alpha-hydroxysteroid dehydrogenase (from rat liver) were unaffected by pyrazole 3. Dead end inhibition studies indicate an ordered binding of cofactor prior to substrate or pyrazole inhibitor.     

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.     

Potent inhibition of the hypothalamic progesterone 5 alpha-reductase by a 5 alpha-dihydroprogesterone analog

The ability of the 5 alpha-dihydroprogesterone analog, 4-aza-4-methyl-5 alpha-pregnane-3,20-dione (AMPD), to inhibit the progesterone 5 alpha-reductase and the two 5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase activities (NADH- and NADPH-linked) from female rat hypothalamus has been studied. Dose response experiments indicate that AMPD is a potent antagonist of hypothalamic progesterone 5 alpha-reduction but is an ineffective inhibitor of the NADPH- and NADH-linked 3 alpha-hydroxysteroid oxidoreductase activities, even at concentrations up to 10 microM. Kinetic analyses of the interaction of AMPD with the progesterone 5 alpha-reductase show that it is a competitive inhibitor versus progesterone (Ki(slope) = 6.2 +/- 0.5 nM; apparent Km (progesterone) = 130 +/- 12 nM) and an uncompetitive inhibitor versus NADPH (Ki(intercept) = 11.8 +/- 0.8 nM). These inhibition patterns are consistent with the concept that NADPH binding precedes that of either AMPD or progesterone. The inhibition of the progesterone 5 alpha-reductase by AMPD does not appear irreversible since preincubation of the enzymatic activity (at 37 degrees C) with inhibitor and NADPH, for periods of time up to 60 min, does not lead to a time-dependent loss of activity. Furthermore, this inhibition can be easily removed via dilution, even following a 60-min preincubation with AMPD and NADPH. It is postulated that the specific and powerful inhibition of the progesterone 5 alpha-reductase by AMPD may be due to this compound functioning as a transition state analog. This inhibitor should prove valuable in studying the characteristics of the progesterone 5 alpha-reductase and the function of hypothalamic progestin metabolism.     

Identification of 7(8) and 8(9) unsaturated adrenal steroid metabolites produced by patients with 7-dehydrosterol-delta7-reductase deficiency (Smith-Lemli-Opitz syndrome)

Patients with Smith-Lemli-Opitz syndrome have impaired ability to synthesize cholesterol due to attenuated activity of 7-dehydrosterol-delta(7)-reductase which catalyses the final step in cholesterol synthesis. Accumulation of 7- and 8-dehydrocholesterol is a result of the disorder and potentially these sterols could be used as precursors of a novel class of delta(7) and delta(8) unsaturated adrenal steroids and their metabolites. In this study, we have analyzed urine from SLOS patients in the anticipation of characterizing such metabolites. Gas chromatography/mass spectrometry (GC/MS) was used in the identification of two major metabolites as 7- and 8-dehydroversions of the well-known steroid pregnanetriol. Other steroids, such as 8-dehydro dehydroepiandrosterone (8-dehydro DHEA) and 7- or 8-dehydroandrostenediol were also identified, and several more steroids are present in urine but remain uncharacterized. As yet, the study provides no evidence for the production of ring-B unsaturated metabolites of complex steroids, such as cortisol. We believe that the following transformations can utilize ring-B dehydroprecursors: StAR transport of cholesterol, p450 side chain cleavage, 17-hydroxylase/17,20-lyase, 3beta-hydroxysteroid dehydrogenase, 3alpha-hydroxysteroid dehydrogenase, 17beta-hydroxysteroid dehydrogenase, 20alpha-hydroxysteroid dehydrogenase and 5beta-reductase. We have yet to prove the activity of adrenal 21-hydroxylase, 11beta-hydroxylase or 5alpha-reductase towards 7- or 8-dehydroprecursors.     

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.     

Inhibition of mitochondrial alpha-ketoglutarate dehydrogenase by 1-methyl-4-phenylpyridinium ion

Effects of 1-methyl-4-phenylpyridinium ion (MPP+) on the activities of NAD+- or NADP+-linked dehydrogenases in the TCA cycle were studied using mitochondria prepared from mouse brains. Activities of NAD+- and NADP+-linked isocitrate dehydrogenases, NADH- and NADPH-linked glutamate dehydrogenases, and malate dehydrogenase were little affected by 2 mM of MPP+. However, alpha-ketoglutarate dehydrogenase activity was significantly inhibited by MPP+. Kinetic analysis revealed a competitive type of inhibition. Inhibition of alpha-ketoglutarate dehydrogenase may be one of the important mechanisms of MPP+-induced inhibition of mitochondrial respiration, and of neuronal degeneration.     

Characterization of NADP+: 3 beta-hydroxysteroid dehydrogenase from microsomes of rat liver

A NADP(+)-dependent 3 beta-hydroxysteroid dehydrogenase activity was localized in the microsomal fraction of rat liver. This enzyme was solubilized and separated completely from 3 alpha-hydroxysteroid dehydrogenase by Matrex red A column chromatography. Partially purified 3 beta-hydroxysteroid dehydrogenase catalyzed the oxidation and reduction between the 3 beta-hydroxyl and 3-ketonic group of steroids or bile acids having no double bond in the A/B ring, but was inactive toward 3 alpha-hydroxyl group. The enzyme required NADP+ for oxidation and NADPH for reduction. The activity was inhibited by p-chloromercuribenzoic acid or p-chloromercuribenzenesulfonic acid at the concentration of 10(-4) M. The molecular weight of the enzyme was estimated to be about 43,000 by Sephadex G-200 column chromatography. From these results, it is concluded that the enzyme is a new type of microsomal NADP+:3 beta-hydroxysteroid dehydrogenase.     

Metabolism of androgens in human hyperplastic prostate: evidence for a differential localization of the enzymes involved in the metabolism

The subcellular distribution of 5 alpha-reductase, 17 beta-hydroxy steroid dehydrogenase, 3 alpha- and 3 beta-hydroxysteroid dehydrogenase activities was studied in human hyperplastic prostate. 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase activities are located in the nuclear envelope. 3 alpha-hydroxysteroid dehydrogenase activity was almost equally distributed between cytosol and membranes, 3 beta-hydroxysteroid dehydrogenase activity was linked to all membranes. Direct testosterone metabolism (transformation into its active metabolite 5 alpha-DHT and into androstenedione, an inactive androgen) takes place only in the nucleus whereas indirect metabolism takes place mainly in the cytoplasm. These findings add new evidence for the mechanism of action of testosterone in prostatic tissue. Testosterone diffuses into the cell, migrates toward the nucleus and is transformed at the nuclear envelope level into two metabolites, DHT and androstenedione. After transformation into its active form, the hormone enters the nucleus whereas the inactive form is released into the cytoplasm. This metabolism could be seen as a control of the amount of active hormone entering the nucleus and being able to bind the androgen receptor.     

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.     

Ontogeny of testicular steroid dehydrogenase enzymes in pig (3 alpha/beta-, 20 alpha- and 20 beta-): evidence for two forms of 3 alpha/beta-hydroxysteroid dehydrogenase.

Three enzymatic activities (3 alpha/beta-hydroxysteroid dehydrogenase, 20 beta- and 20 alpha-hydroxysteroid dehydrogenases) were measured in testes of pigs as a function of age. Earlier studies reported a highly purified 20 beta-hydroxysteroid dehydrogenase from neonatal pig testes that also showed strong 3 alpha/beta-hydroxysteroid dehydrogenase activity [Ohno et al., J. Steroid Biochem. Molec. Biol. 38 (1991) 787-794]. We report here that neonatal pigs testis is rich in 3 alpha/beta- and 20 beta-hydroxysteroid dehydrogenase activities, both of which fall to low levels (measured as specific activity) at 60 days. Thereafter the activity of 3 alpha/beta-reduction rises to high levels whereas 20 beta-reduction remains low. Activity of 20 alpha-reduction is of intermediate level in the neonate, falls to a nadir at 60 days and rises to high levels in the mature animal. Western blots of cytosolic proteins show that the bifunctional enzyme (3 alpha/beta-plus 20 beta-hydroxysteroid dehydrogenase) is high in neonatal testes and falls to low levels at maturity. It is proposed that the neonatal testis possesses the bifunctional enzyme which is replaced by a second enzyme at maturity, that is a 3 alpha/beta-hydroxysteroid dehydrogenase without 20 beta-reductase activity. The possible functional significance of these changes is considered.     

Delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in canine pancreas

Activity of delta 5-3 beta-hydroxysteroid dehydrogenase coupled with steroid-delta 5-4-isomerase was demonstrated for the first time in the pancreas. The enzyme complex was assayed by measuring the conversion of pregnenolone to progesterone as well as of dehydroepiandrosterone to androstenedione and found to be localized primarily in the mitochondrial fraction of dog pancreas homogenates. The delta 5-3 beta-hydroxysteroid dehydrogenase used either NAD+ or NADP+ as co-substrates, although maximal activity was observed with NAD+. In phosphate buffer, pH 7.0 and 37 degrees C, the apparent Km values of the dehydrogenase were 6.54 +/- 0.7 microM for pregnenolone and 9.61 +/- 0.8 microM for NAD+. The apparent Vmax was determined as 0.82 +/- 0.02 nmol min-1 mg-1. Under the same conditions the Km values for dehydroepiandrosterone and NAD+ were 3.3 +/- 0.2 microM and 9.63 +/- 1.6 microM, respectively, and the apparent Vmax was 0.62 +/- 0.01 nmol min-1 mg-1.     

Inhibition of 3(17)beta-hydroxysteroid dehydrogenase from Pseudomonas testosteroni by steroidal A ring fused pyrazoles

Several 2,3- and 3,4-steroidal fused pyrazoles have been investigated as potential inhibitors of NAD(P)H-dependent steroid oxidoreductases. These compounds are proven to be potent, specific inhibitors for 3(17) beta-hydroxysteroid dehydrogenase from Pseudomonas testosteroni with Ki values of 6-100 nM. In contrast, the activities of 3 alpha,20 beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans, steroid 5 alpha-reductase from rat prostate, and 3 alpha-hydroxysteroid dehydrogenase from rat liver were unaffected by micromolar concentrations of these compounds. Product and dead-end inhibition studies indicate an ordered association to the beta-dehydrogenase with the cofactor binding prior to substrate or inhibitor. From the results of double inhibition experiments, it is proposed that inhibition occurs through formation of an enzyme-NAD+-inhibitor ternate. On the basis of pH profiles of Vm/Km, Vm, and 1/Ki and of absorbance difference spectra, a hypothetical mechanism of inhibition by the steroidal pyrazoles, drawn by analogy from the inhibition of liver alcohol dehydrogenase by alkylpyrazoles [Theorell, H., & Yonetani, T. (1963) Biochem. Z. 338, 537-553; Andersson, P., Kvassman, J. K., Lindstr?m, A., Oldén, B., & Pettersson, G. (1981) Eur. J. Biochem. 113, 549-554], is reconsidered. The pH studies and enzyme modification experiments by diethyl pyrocarbonate suggest the involvement of histidine in binding of the inhibitor. A modified proposal for the structure of the enzyme-NAD+-steroidal pyrazole complex is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of two dihydrodiol dehydrogenases associated with 3(17)alpha-hydroxysteroid dehydrogenase activity in mouse kidney

Dihydrodiol dehydrogenase activity was detected in the cytosol of various mouse tissues, among which kidney exhibited high specific activity comparable to the value for liver. The enzyme activity in the kidney cytosol was resolved into one major and three minor peaks by Q-Sepharose chromatography: one minor form cross-reacted immunologically with hepatic 3 alpha-hydroxysteroid dehydrogenase and another with aldehyde reductase. The other minor form was partially purified and the major form was purified to homogeneity. These two forms, although different in their charges, were monomeric proteins with the same molecular weight of 39,000 and had similar catalytic properties. They oxidized cis-benzene dihydrodiol and alicyclic alcohols as well as trans-dihydrodiols of benzene and naphthalene in the presence of NADP+ or NAD+, and reduced several xenobiotic aldehydes and ketones with NAD(P)H as a cofactor. The enzymes also catalyzed the oxidation of 3 alpha-hydroxysteroids and epitestosterone, and the reduction of 3- and 17-ketosteroids, showing much lower Km values (10(-7)-10(-6) M) for the steroids than for the xenobiotic alcohols. The results of mixed substrate experiments, heat stability, and activity staining on polyacrylamide gel electrophoresis suggested that, in the two enzymes, both dihydrodiol dehydrogenase and 3(17)alpha-hydroxysteroid dehydrogenase activities reside on a single enzyme protein. Thus, dihydrodiol dehydrogenase existed in four forms in mouse kidney cytosol, and the two forms distinct from the hepatic enzymes may be identical to 3(17)alpha-hydroxysteroid dehydrogenases.     

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.     

Adrenal 11-beta hydroxysteroid dehydrogenase activity in response to stress

This work studied the effect of stresses produced by simulated gavage or gavage with 200 mmol/L HCl two hours before adrenal extraction, on the activities of the 11beta-hydroxysteroid dehydrogenase 1 and 11beta-hydroxysteroid dehydrogenase 2 isoforms present in the rat adrenal gland. These activities were determined on immediately prepared adrenal microsomes following incubations with 3H-corticosterone and NAD+ or NADP+. 11-dehydrocorticosterone was measured as an end-product by TLC, and controls were adrenal microsomes from rats kept under basal (unstressed) conditions. 11beta-hydroxysteroid dehydrogenase 1 activity, but not 11beta-hydroxysteroid dehydrogenase 2 activity, was increased under both stress-conditions. Homeostatically, the stimulation of 11beta-hydroxysteroid dehydrogenase 1 activity would increase the supply of glucocorticoids. These, in turn, would activate the enzyme phenylethanolamine N-methyl transferase, thereby improving the synthesis of epinephrine as part of the stress-response.