Hydroxysteroid Oxidoreductase Research Articles

Progesterone Exerts a Neuromodulatory Effect on Turning Behavior of Hemiparkinsonian Male Rats: Expression of 3 α -Hydroxysteroid Oxidoreductase and Allopregnanolone as Suggestive of GABAA Receptors Involvement.

There is a growing amount of evidence for a neuroprotective role of progesterone and its neuroactive metabolite, allopregnanolone, in animal models of neurodegenerative diseases. By using a model of hemiparkinsonism in male rats, injection of the neurotoxic 6-OHDA in left striatum, we studied progesterone's effects on rotational behavior induced by amphetamine or apomorphine. Also, in order to find potential explanatory mechanisms, we studied expression and activity of nigrostriatal 3α-hydroxysteroid oxidoreductase, the enzyme that catalyzes progesterone to its active metabolite allopregnanolone. Coherently, we tested allopregnanolone for a possible neuromodulatory effect on rotational behavior. Also, since allopregnanolone is known as a GABAA modulator, we finally examined the action of GABAA antagonist bicuculline. We found that progesterone, in addition to an apparent neuroprotective effect, also increased ipsilateral expression and activity of 3α-hydroxysteroid oxidoreductase. It was interesting to note that ipsilateral administration of allopregnanolone reversed a clear sign of motor neurodegeneration, that is, contralateral rotational behavior. A possible GABAA involvement modulated by allopregnanolone was shown by the blocking effect of bicuculline. Our results suggest that early administration of progesterone possibly activates genomic mechanisms that promote neuroprotection subchronically. This, in turn, could be partially mediated by fast, nongenomic, actions of allopregnanolone acting as an acute modulator of GABAergic transmission.

Antiproliferative effects of flavonoid fractions from Calendula officinalis flowers in parent and tamoxifen resistant T47D human breast cancer cells

The risk of human breast cancer is concerned to cumulative exposure of the breast cells to endogenous estrogens. Strategies aiming at reducing the production of estrogens may be useful for the prevention of estrogens-related breast cancer. Several natural products with plant origin have the potential value as chemo-preventive or therapeutic agents in cancer. Flavonoids, the natural polyphenol compounds, have shown antiviral, anti-inflammatory, anti-mutagenic and anti-carcinogenic activities. Calendula officinalis, a member of compositae family, is well known for its pharmacological effects such as anti-inflammatory, anti-viral, anti-HIV, anti-tumor, anti-mutagenic and cytotoxic properties. Mechanism of the possible chemo-preventing action of flavonoids has not been yet completely understood. They may exert their chemo-preventive and anti-carcinogenic effects in estrogen-dependent cells by inhibiting aromatase, 17?-hydroxysteroid oxidoreductase and other enzymes involved or by possessing estrogenic and anti-estrogenic activities. In this study, three major flavonoid fractions were separated from a methanol extract of Calendula officinalis flowers by preparative TLC. These fractions were evaluated for inhibition of parent and tamoxifen resistant T47D human breast cancer. The fractions did not have inhibitory effect. We also examined the effect of quercetin and isorhamnetin on the growth of parent and resistant T47D cells in the presence or absence of tamoxifen. It was found that quercetin increased cell proliferation of the resistant T47D cells at the presence of tamoxifen but no effect was detected by using quercitin itself. In consequence, isorhamnetin had not any proliferative or anti-proliferative activity on the both cell lines. Many mechanisms can contribute to the affects obtained on the cell growth.

Activity and expression of progesterone metabolizing 5α-reductase, 20α-hydroxysteroid oxidoreductase and 3α(β)-hydroxysteroid oxidoreductases in tumorigenic (MCF-7, MDA-MB-231, T-47D) and nontumorigenic (MCF-10A) human breast cancer cells

BackgroundRecent observations indicate that human tumorous breast tissue metabolizes progesterone differently than nontumorous breast tissue. Specifically, 5α-reduced metabolites (5α-pregnanes, shown to stimulate cell proliferation and detachment) are produced at a significantly higher rate in tumorous tissue, indicating increased 5α-reductase (5αR) activity. Conversely, the activities of 3α-hydroxysteroid oxidoreductase (3α-HSO) and 20α-HSO enzymes appeared to be higher in normal tissues. The elevated conversion to 5α-pregnanes occurred regardless of estrogen (ER) or progesterone (PR) receptor levels. To gain insight into these differences, the activities and expression of these progesterone converting enzymes were investigated in a nontumorigenic cell line, MCF-10A (ER- and PR-negative), and the three tumorigenic cell lines, MDA-MB-231 (ER- and PR-negative), MCF-7 and T-47D (ER- and PR-positive).MethodsFor the enzyme activity studies, either whole cells were incubated with [14C]progesterone for 2, 4, 8, and 24 hours, or the microsomal/cytosolic fraction was incubated for 15–60 minutes with [3H]progesterone, and the metabolites were identified and quantified. Semi-quantitative RT-PCR was employed to determine the relative levels of expression of 5αR type1 (SRD5A1), 5αR type 2 (SRD5A2), 20α-HSO (AKR1C1), 3α-HSO type 2 (AKR1C3), 3α-HSO type 3 (AKR1C2) and 3β-HSO (HSD3B1/HSD3B2) in the four cell lines using 18S rRNA as an internal control.ResultsThe relative 5α-reductase activity, when considered as a ratio of 5α-pregnanes/4-pregnenes, was 4.21 (± 0.49) for MCF-7 cells, 6.24 (± 1.14) for MDA-MB-231 cells, 4.62 (± 0.43) for T-47D cells and 0.65 (± 0.07) for MCF-10A cells, constituting approximately 6.5-fold, 9.6-fold and 7.1 fold higher conversion to 5α-pregnanes in the tumorigenic cells, respectively, than in the nontumorigenic MCF-10A cells. Conversely, the 20α-HSO and 3α-HSO activities were significantly higher (p < 0.001) in MCF-10A cells than in the other three cell types. In the MCF-10A cells, 20α-HSO activity was 8-14-fold higher and the 3α-HSO activity was 2.5-5.4-fold higher than in the other three cell types. The values of 5αR:20α-HSO ratios were 16.9 – 32.6-fold greater and the 5αR:3α-HSO ratios were 5.2 – 10.5-fold greater in MCF-7, MDA-MB-231 and T-47D cells than in MCF-10A cells. RT-PCR showed significantly higher expression of 5αR1 (p < 0.001), and lower expression of 20α-HSO (p < 0.001), 3α-HSO2 (p < 0.001), 3α-HSO3 (p < 0.001) in MCF-7, MDA-MB-231 and T-47D cells than in MCF-10A cells.ConclusionThe findings provide the first evidence that the 5αR activity (leading to the conversion of progesterone to the cancer promoting 5α-pregnanes) is significantly higher in the tumorigenic MCF-7, MDA-MB-231 and T-47D breast cell lines than in the nontumorigenic MCF-10A cell line. The higher 5αR activity coincides with significantly greater expression of 5αR1. On the other hand, the activities of 20α-HSO and 3α-HSO are higher in the MCF-10A cells than in MCF-7, MDA-MB-231 and T-47D cells; these differences in activity correlate with significantly higher expression of 20α-HSO, 3α-HSO2 and 3α-HSO3 in MCF-10A cells. Changes in progesterone metabolizing enzyme expression (resulting in enzyme activity changes) may be responsible for stimulating breast cancer by increased production of tumor-promoting 5α-pregnanes and decreased production of anti-cancer 20α – and 3α-4-pregnenes.

Inhibition of 17 -Hydroxysteroid Oxidoreductase by Flavonoids in Breast and Prostate Cancer Cells

Several flavonoids and isoflavonoids were found to inhibit 17beta-oxidoreduction of estrogens by the purified 17beta-HSOR type 1, or in cell lines expressing 17beta-HSOR type 1 enzyme (T-47D breast cancer cells) or type 2 (PC-3 prostate cancer cells). The structural demands for the inhibition of estrone (E1) reduction and estradiol (E2) oxidation catalyzed by 17beta-HSOR types 1 and 2, respectively, were not identical. Flavones, flavanones, and isoflavones hydroxylated at both the double ring (positions 5 and 7) and ring B (position 4') were the most potent inhibitors of E1 reduction in T-47D cells, and by the purified type 1 enzyme whereas flavones hydroxylated at positions 3, 5, and 7 of rings A and C, with or without a hydroxyl group in ring B, were capable of inhibiting E2 oxidation in PC-3 cells. Change to flavanone structure, or hydroxylation at position 3 of ring C of flavones, or methylation of the hydroxyl group at position 4' of ring B of flavones and isoflavones reduced or abolished their inhibitory activity on E1 reduction in T-47D cells. On the contrary, hydroxyl group at position 3 of flavones (flavonol structure) markedly increased the inhibition of E2 oxidation in PC-3 cells. Thus, changes in the number and location of hydroxyl groups may discriminate inhibition of E1 reduction and E2 oxidation. Some of the differences may be due to differences in pharmacokinetics of these compounds in T-47D and PC-3 cells. Inhibition of 17beta-HSORs could lead to an alteration in the availability of the highly active endogenous estrogen, but the effects of these compounds in vivo cannot be predicted on the basis of these results alone. Some of these compounds (isoflavones) are estrogenic per se, and they may replace endogenous estrogens, whereas flavones are only very weakly estrogenic or nonestrogenic. Regarding prevention or treatment of estrogen-related diseases, apigenin, coumestrol, and genistein raise special interest.

Steroid metabolism in the hormone dependent MCF-7 human breast carcinoma cell line and its two hormone resistant subpopulations [formula omitted] and [formula omitted

Androgen and estrogen metabolism was investigated in the hormone-dependent human breast cancer cell line MCF-7 and its two hormone-resistant sublines MCF-7 LCC1 and MCF-7 LCC2 . Using the product isolation method, the activity of aromatase, 5α-reductase, 3α β - hydroxysteroid oxidoreductase and 17β-hydroxysteroid oxidoreductase were investigated isolating the following steroids: estriol (E 3), estradiol (E 2), estrone (E 1), 3α β - androstanediol (A-diol), testosterone (T), dihydrotestosterone (DHT), androsterone (AND), androstenedion (4-AD) and androstanedione (A-dion). For all experiments, cells were preincubated with cortisol and subsequently incubated with [ 14C]T or [ 14C]4-AD as the substrate in medium without phenol red and with serum charcoal stripped of steroids. The results showed no aromatase activity in any of the cell lines under the experimental conditions used, and preincubation with cortisol had no effect on the enzyme activity. With [ 14C]T as the substrate, the metabolized level of DHT was very similar in the three cell lines, though MCF-7 LCC1 and MCF-7 LCC2 utilized the substrate to a much lesser extent. The amount of DHT and 4-AD produced were comparable in the two hormone-resistant cell lines, while the amount of 4-AD was significantly higher in MCF-7 cells. No differences in enzyme activity were found in the three cell lines when [ 14C]4-AD was used as the substrate. This study showed an altered androgen metabolism in the MCF-7 LCC1 and MCF-7 LCC2 sublines compared to the parent MCF-7. However, since treatment with DHT and T inhibited cell growth equally well in all three tumor cell lines, it is unlikely that the found differences in steroid metabolism was involved in the acquisition of the endocrine resistance of the two MCF-7 sublines.

Distribution of estradiol‐17β hydroxysteroid oxidoreductase in the urogenital tract of control and neonatally estrogenized male mice: Immunohistochemical, enzymehistochemical, and biochemical study

The present study was conducted to investigate the regional distribution of the enzymes catalyzing the interconversion of the hydroxyl and carbonyl groups at C-17 of the estrogen molecule within the male urogenital tract of adult mouse and to test the hypothesis, whether regional differences in the distribution are critical for estrogen responses. The highest ratios of NADPH-dependent 3H-estrogen reduction to oxidation at C-17 of cell-free homogenates were obtained from coagulating gland and seminal vesicle as well as from the prostatic and lower intrapelvic urethra, which are considered the most estrogen-sensitive parts of the male urogenital tract. Both NADP- and NAD-dependent oxidation of 3H-17 beta-estradiol were low or nondetectable at these sites. The epithelium of the lower and prostatic urethra as well as the periurethral collecting ducts were stained with the antibody prepared against human placental 17 beta-hydroxysteroid oxidoreductase. The NAD-dependent 3H-estradiol-17 beta oxidase activity was highest in the bladder epithelium, and the activity declined sharply in the urinary tract from the bladder downward. The lowest detectable activities were found in vas deferens and prostate (combined ventral and dorsolateral lobes). The uneven distribution of estradiol-17 beta oxidase activity may provide additional explanation for the regional differences of estrogen responses. The NADPH-dependent 17 beta-reduction of estrone and the immunohistochemical staining of the human placental estradiol-17 beta oxidoreductase antigenicity were not significantly altered after neonatal estrogenization. These findings do not lend any support to the idea that the increased estrogen sensitivity observed after neonatal estrogenization is associated with changes in 17 beta-oxidoreduction. However, the possibility remains that there are specific sites (e.g., epithelium of prostatic urethra and collecting ducts) in which the changes in 17 beta-oxidoreduction of estrogen does play a role in the regulation of estrogen action.

Pyridine Nucleotide Levels under Conditions of 5α-Dihydrotestoster one-Stimulated 17β-Estradiol Formation from Estrone and Pathway of Nicotinamide Adenine Dinucleotide Biosynthesis in Placental Villiin Vitro*

It has been reported that testosterone, 5 alpha-dihydrotestosterone and 20 alpha-dihydroprogesterone, substrates for pyridine nucleotide-dependent hydroxysteroid oxidoreductases, stimulate the conversion of estrone to 17 beta-estradiol (E2) by placental villi in vitro. Their enzyme-catalyzed oxidation generates either NADH or NADPH. If the concentration of either reduced nucleotide were rate determining in the conversion of estrone to E2, then increases in NADH or NADPH levels as the result of steroid oxidation could stimulate E2 formation. In this investigation, enzymatic assays were used to quantitate NAD, NADP, NADH, and NADPH in villous tissue from term placenta under conditions where E2 formation was stimulated by 5 alpha-dihydroxytestosterone. On the basis of concurrent observations that NAD levels varied initially and decreased in tissue samples in culture over a 24-h period, the ability of villi to incorporate [14C]nicotinic acid or [14C]nicotinamide into NAD was also examined. No changes were detected in the ratios of oxidized to reduced [14C]nicotinamide nucleotides under stimulatory conditions. NAD was formed only from nicotinic acid. The data suggest that NAD and NADP reduction, if it is the basis for stimulation, is tightly coupled to reoxidation. It would also appear that media used widely for villous tissue and cell culture may not be optimal for pyridine nucleotide synthesis.

Effect of Aging on Kinetic Parameters of 3α(β)-Hydroxysteroid Oxidoreductases in Epithelium and Stroma of Human Normal and Hyperplastic Prostate

The accumulation of 5 alpha-dihydrotestosterone (DHT), particularly in stroma, is a possible etiological factor in regard of the age-dependent development of benign prostatic hyperplasia (BPH). In this context, we have recently demonstrated age-dependent alterations of 5 alpha-reductase, which is responsible for the irreversible conversion of testosterone to DHT. Therefore, it was also of interest to study possible age-dependent alterations of those enzymes mainly involved in the reversible metabolism of DHT to 5 alpha-androstanediols. Thus, we determined, in the presence of NADPH/NADP+, kinetic parameters [Km and maximum velocity (Vmax)] of 3-hydroxysteroid oxidoreductases (3 alpha-HSORred, 3 beta-HSORred, and 3 alpha-HSORox) in separated epithelium and stroma of 10 normal (NPR) and 20 hyperplastic prostates (BPH) and correlated the data with the age of the donors (15-86 yr). The mean Km (nanomolar concentrations +/- SEM) of 3 alpha-HSORred was significantly (P less than 0.01) higher in epithelium (NPR, 1391 +/- 181; BPH, 2150 +/- 157) than in stroma (NPR, 778 +/- 22; BPH, 749 +/- 62), indicating the presence of epithelial and stromal enzymes. The mean Km values of 3 beta-HSORred and 3 alpha-HSORox were similar. Concerning 3 alpha-HSORred, the mean potential capacity, i.e. the quotient of Vmax/Km (+/- SEM), was significantly (P less than 0.01) higher in epithelium (0.56 +/- 0.08) than in stroma (0.19 +/- 0.02) of NPR, while in BPH nearly identical mean potential capacities were found in epithelium (0.33 +/- 0.04) and stroma (0.26 +/- 0.02). The respective Vmax/Km of 3 beta-HSORred and 3 alpha-HSORox were significantly (P less than 0.05) lower. In addition, the potential capacity of all three enzymes was distinctly lower than the potential DHT-forming capacity of 5 alpha-reductase. With advancing age, the Vmax/Km decreased significantly (P less than 0.001; 3 alpha-HSORred and 3 beta-HSORred) or tendentiously (3 alpha-HSORox) in epithelium, while in stroma a significant (P less than 0.001; 3 alpha-HSORred and 3 alpha-HSORox) or tendentious (3 beta-HSORred) increase with age was found. Our results indicate that aging has a significant impact on DHT-removing enzymes. However, these enzymes counterbalance only in part the strong potential capacity of 5 alpha-reductase.

Regional distribution of microsomal xenobiotic and steroid metabolism in kidney microsomes from rainbow trout

The regional distribution of microsomal cytochrome P-450-mediated reactions with exogenous and endogenous substrates in the kidney of rainbow trout was studied. The cytochrome P-450-dependent 7-ethoxyresorufin- and 7-ethoxycoumarin-0-deethylase activities were significantly higher (3-4 and 10-14 fold, respectively) in the trunk kidney than in the head kidney, whereas ethylmorphine-N-demethylase activity was evenly distributed along the kidney. The microsomal cytochrome P-450-dependent steroid hydroxylases and steroid reducing enzymes when using androstenedione as substrate also exhibited a regional distribution in trout kidney. The 6β- and 16-hydroxylase activities as well as the 5α-reductase and 17 hydroxysteroid oxidoreductase activities were higher in the anterior part of the trunk kidney than in the head kidney and posterior trunk kidney.

Occurrence of hydroxysteroid oxidoreductases in liver of turtles

1. 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. 2. The following enzymes were detected (substrates in brackets): 3α-hydroxysteroid oxidoreductase (androsterone), 3β-hydroxysteroid oxidoreductase (DHEA) and 17β-hydroxysteroid oxidoreductase (testosterone, oestradiol-17β). NAD as well as NADP were effective as cofactors. 3. 3. In fresh-water turtle, highest activities of the 3α-enzyme were measured in the 20% AS fraction (cofactor NAD), of the 3β-enzyme in the 60% AS fraction (cofactor NAD) and of the 17β-enzyme in the 40% AS fraction (cofactor NADP). 4. 4. In sea-water turtle, highest activities were observed for all three enzymes in the 60% AS fraction. 5. 5. Generally, enzyme activities were higher in sea-water turtles. The most active enzyme in both turtles was found to be the 3α-hydroxysteroid oxidoreductase, followed by the 17β- and the 3β-hydroxysteroid oxidoreductases.

Biotransformation of 3‐methoxy‐8,14‐seco‐1,3,5(10),9(11) estratetraen‐14,17‐dione (secodione) to its 17β‐hydroxy derivative (secol) using immobilized yeast cells (Pichia farinosa Y‐118)

The yeast cells of Pichia farinosa Y-118 were immobilized in polyacrylamide gel and used for 17 beta-oxidoreduction of secondione to secol. The loss of hydroxysteroid oxidoreductase activity of cells was found to be insignificant during immobilization. The preparation exhibited greater temperature stability as compared to free cells. The ratio of reaction volume to the volume of immobilized biocatalyst in the range 1.4-1.9 was found to be satisfactory for the reaction conditions studied. This ratio played a significant role in the stability of the catalyst particle, since beyond a critical value the disintegration of gel granules was rapid resulting in sharp decline of activity. The immobilized cell preparation could be used 50 times over a period of 100 days without loss of activity. However, the activity declined in further reuses, leaving the preparation 50 and 35% active after its 60th and 70th uses, respectively.

Steroid sulfatase and 17β-hydroxysteroid oxidoreductase activities in mouse tissues

The metabolism of estrone sulfate and dehydroisoandrosterone sulfate to the free, unconjugated steroids, estrone and dehydroisoandrosterone, was demonstrated in more than thirty different tissues from male and female BALB/c mice. The activity of steroid sulfatase, when expressed per mg tissue, was greatest in both the pituitary gland and the adrenal glands. The pituitary gland, however, had the lowest capacity for hydrolysis of steroid sulfates while the liver had the greatest capacity. 17β-Hydroxysteroid oxidoreductase activity also was demonstrated in all mouse tissues by the formation of estradiol-17β when using estrone sulfate as the substrate. The highest apparent activity for 17β-hydroxysteroid oxidoreductase was found in lung tissue, and the greatest capacity to form estradiol-17β from estrone sulfate was found in liver, lungs, kidneys and testes. This study demonstrates that the majority of mouse tissues have steroid sulfatase and l 7β -hydroxysteroid oxidoreductase activities.

Estrogen metabolism in neural tissues of rabbits: 17 beta - hydroxysteroid oxidoreductase activity.

Incubation of adult rabbit neural tissue homogenates with (3H)-estrone and (3H)-estradiol revealed that the conversion of estrone to estradiol is higher in both male and female animals than estradiol to estrone. Both 17 oxidation and reduction are higher in male animals than in females. However, it is observed that the quotient of estrone leads to estradiol/estradiol leads to estrone for pituitary tissue and hypothalamus are higher in females than in males. There is no such dimorphism in cerebral cortex. The overall metabolism in pituitary is higher than in hypothalamus and cortex in both sexes. These results suggest that 17 beta hydroxysteroid oxidoreductase activity may play an important role in the regulation of estrogen function in neuroendocrine tissues.

Induction of steroid glucuronyltransferase and repression of hydroxysteroid oxidoreductase: Recent findings

Steroid hormones are excreted mainly as glucuronides. Malfunction or deficiency of the enzyme which glucuronidates steroid hormones (UDP-glucuronate-17β-oestradiol 3-glucuronyltransferase) is relatively rare. On the other hand, several studies have shown that the enzyme is inducible. One major objection to these induction studies is that they have been carried out with nonphysiological substrates. Therefore, the kinetics and physicochemical properties of the enzyme from various sources have now been investigated using physiological substrates. In view of the extensive use of steroid hormones in medicine, it was important to know whether steroid glucuronyltransferases are induced by their own substrates. The results showed that ovariectomy leads to a 56% decrease in oestrone glucuronyltransferase activity. Administration of oestradiol-17β to rats induced the enzyme to normal activity after 12 days; after 15 days, the activity was twice the control value. Actinomycin d and cycloheximide blocked the oestradiol-17β-induced increase in enzyme activity. These results provide strong evidence that oestrogen glucuronyltransferase is induced by its substrate. It is well established that hydroxysteroid oxidoreductases (HSOR) play a major role in the intermediary metabolism of androgens and oestrogens. Previously, it was shown that the activity of the 17β-HSOR, catalysing the oxidoreduction of oestradiol-17β and oestrone in rat liver, is different in female and male animals. When female animals were gonadectomised, the time course of development of enzyme activity was almost identical to that observed in male rats. This finding indicated that the female hormone may have a repressive effect on the activity of the 17β-HSOR in adult rats. Further studies revealed that isoenzymes exist in the cytosol fraction, two of which show a clear dependence on the hormonal status of the rat. With NAD as cofactor, the apparent Michaelis constant ( k m ) of the major 17β-HSOR from female rats was found to be 514 μmol/l, whereas the corresponding value for male rats was considerably lower at 58 μmol/l. With NADP as cofactor, the apparent k m values of the female and the male enzyme were similar. These results indicate that oestrogens regulate the activity of the soluble 17β-HSOR in liver by different mechanisms, including one where oestrogens have a “repressing” effect on enzyme synthesis.

Cellular Regulation of the Metabolism of Androgens in Rat Oral Mucosa: I. Effect of Medroxyprogesterone Acetate (MPA) on the Soluble Supernatant (Cytosol) Activating Microsomal Δ4-3-Ketosteroid-5α-Reductase and 17β -Hydroxysteroid Oxidoreductase Enzyme Systems

Soluble supernatant (cytosol) of rat oral mucosa was found to contain a factor accelerating the activity of microsomal delta4-3-ketosteroid-5alpha-reductase and 17beta-hydroxysteroid oxidoreductase enzymes metabolizing testosterone. Systemic administration of medroxyprogesterone acetate further increased this cytosolic activity.

Effects of 2-mercaptoethanol and aging [formula omitted] on 17β -hydroxysteroid oxidoreductase of guinea pig liver microsomes

When microsomes were prepared in 2-mercaptoethanol V max for 17β -hydroxysteroid oxidoreductase(17β -HSD) was greater, the K m for NAD+ was greater and the K m for testosterone lower than in its absence. During storage at 4δ V max increased in the presence of 2-mercaptoethanol and decreased in its absence; K m values for testosterone and NAD + increased during storage in both cases. The presence or absence of 2-mercaptoethanol did not affect the extent or time-course of inactivation of 17β -HSD by trypsin or phospholipase A. Furthermore, no differences were detected in sedimentation properties on sucrose density gradients suggesting that the differences and changes in the kinetic behavior of 17β -HSD reflect a conformational flexibility at the active site and are not due to extensive changes in the structure of the microsomes. 17β -HSD exposed to 2-mercaptoethanol was subject to substrate inhibition by testosterone, a type of inhibition not previously reported for this enzyme.

NAD- and NADP-dependent 7α-hydroxysteroid dehydrogenases from Bacteroides fragilis

Twenty strains of Bacteroides fragilis were screened for hydroxysteroid oxidoreductase activity in cell-free preparations. Eighteen strains were shown to contain NAD-dependent 7α-hydroxysteroid dehydrogenase. Sixteen of the strains containing the NAD-dependent enzyme also contained NADP-dependent 7α-hydroxysteroid dehydrogenase, but invariably in lesser amounts. A strain particularly rich in both 7α-hydroxysteroid dehydrogenase activities was selected for further study. Measurement of activity as a function of pH revealed a fairly sharp optimal activity range of 9.5–10.0 for the NAD-dependent enzyme and a broad flat optimal range of 7.0–9.0 for the NADP-dependent enzyme. Michaelis constants for trihydroxy-bile acids for the NAD-dependent enzyme were in the range of 0.32–0.34 mM, whereas dihydroxy-bile acids gave a K m of 0.1 mM. Thin-layer chromatography studies on the oxidation product of 3α,7α-dihydroxy-5β-cholanoic acid (chenodeoxycholic acid )by the dehydrogenase revealed a band corresponding to that of synthetic 3α-hydroxy, 7-keto-5β-cholanoic acid. Similarly the oxidation product of chenodeoxycholic acid by both 7α-hydroxysteroid dehydrogenase and commercially available 3α-hydroxysteroid dehydrogenase revealed a band corresponding to that of synthetic 3,7-diketo-5β-cholanoic acid. Neither of these two oxidation products could be distinguished from those by the Escherichia coli dehydrogenase oxidation previously reported. Disc-gel electrophoresis of a cell-free lyophilized preparation indicated one active band for NAD-dependent activity of mobility similar to that for the NADP-dependent E. coli enzyme. The NADP-dependent dehydrogenase was unstable and rapidly lost activity after polyacylamide disc-gel electrophoresis, ultracentrifugation, freezing on refrigeration at 4°C. No 3α- or 12α-oriented oxidoreductase activity was demonstrated in any of the strains examined.

Feminine patterns of androgen metabolism and presence of delta5, 3beta-hydroxysteroid oxidoreductases in target organs of genetic rat male pseudohermaphrodites.

SummaryThe metabolism of dehydroepiandrosterone-7-3H, androstenedione-1,2-3H, and testosterone-1,2-3H by the preputial glands and genital skin (prepuce) was determined in genetic male pseudohermaphrodites, and in their littermate King-X Holtzman males and females. Both tissues in each of the three animals have an active Δ5-3β-hydroxysteroid oxidoreductase system capable of transforming dehydroepiandrosterone to testosterone and testosterone metabolites in a similar fashion to that of the woffian duct and genital tubercle of the fetus of this species as previously reported. The present results also indicate that the pattern of androgen metabolism in both tissues of the rat male pseudohermaphrodites has an elevated amount of 17β-hydroxylated and 3α-hydroxylated 5α-androstane products resembling that of the corresponding tissues of the female and is significantly different from that of its littermate male. Thus, it appears that there is a sex dependent difference in the pattern of androgen metabolizing enzym...

Influence of an 18-hydroxyl group on the interaction of oestrogens and hydroxysteroid oxidoreductases.

1. Partially purified 17beta-hydroxy steroid-NAD(+) oxidoreductases, prepared from Pseudomonas testosteroni (EC 1.1.1.51), human term placenta (EC 1.1.1.62) and the cytoplasmic fraction of rat liver (EC 1.1.1.-) were tested for their ability to catalyse the oxidoreduction of 18-hydroxyoestradiol-17beta and 18-hydroxyoestrone. The products of incubation were identified by chromatographic procedures and by mass spectrometry. 2. The Pseudomonas enzyme catalysed both the oxidation of 18-hydroxyoestradiol-17beta and the reduction of 18-hydroxyoestrone; in contrast, the placental and rat liver enzymes only catalysed the reduction of 18-hydroxyoestrone. 3. These results were confirmed, by using a spectrophotometric assay; equimolar quantities of oestradiol-17beta and 18-hydroxyoestradiol-17beta were oxidized at approximately the same rate by the microbial enzyme. 4. These findings suggest that 18-hydroxyoestradiol-17beta may be a normal oestrogen metabolite. 5. The differences in ability of the mammalian and microbial enzymes to metabolize 18-hydroxylated oestrogens is explained on the basis of recognition sites with different geometrical dimensions, characteristic of the placental (Descomps & Crastes de Paulet, 1969) and the microbial steroid enzymes (Fosset & Crastes de Paulet, 1967).

The 17 β hydroxysteroïd NAD (P) oxidoreductases from female rabbit liver cytosol: separation and characterization

Summary DEAE cellulose chromatography achieved the separation of several 17 β hydroxysteroid NAD (P) oxidoreductase activities from rabbit liver cytosol; two of these activities differ strongly by their anionic character and electrophoretic mobility, as well as by their substrate or coenzyme specificity; the purification of these two fractions (A and C) was performed further. The fraction A contains a weakly anionic 17 β hydroxysteroid oxidoreductase. Its molecular weight is about 25 – 30 000 (sephadex G 100 filtration), both testosterone and estradiol are substrates; NAD or NADP are coenzymes, and the Km for testosterone is 5.10−4 M. From the second fraction (C) was separated a very anionic testosterone NADP oxidoreductase with high affinity for testosterone (Km 3 ± 0,5 10−6 M). The four isozymes shown by polyacrylamide gel electrophoresis are resolved into a single peptid chain of M.W. 36 000 by SDS polyacrylamide gel electrophoresis. The purified enzyme of fraction (C) exhibits a high 3α hydroxysteroid NADP oxidoreductase activity: experimental results are given showing electrophoretic and kinetic evidences for the identity of these two enzymes in fraction C of the adult female rabbit.