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PubMed1990

Characterization and localization of progesterone 5 alpha-reductase from cell cultures of foxglove (Digitalis lanata EHRH).

Progesterone 5 alpha-reductase, which catalyses the reduction of progesterone to 5 alpha-pregnane-3,20-dione, was isolated and characterized from cell cultures of Digitalis lanata (foxglove). Optimum enzyme activity was observed at pH 7.0, and the enzyme had an apparent Km value of 30 microM for its substrate progesterone. The enzyme needs NADPH as reductant, which could not be replaced by NADH. For NADPH, the apparent Km value is 130 microM. The optimum temperature was 40 degrees C; at temperatures below 45 degrees C, the product 5 alpha-pregnane-3,20-dione was reduced by a second reaction to 5 alpha-pregnan-3 beta-ol-20-one. Progesterone 5 alpha-reductase activity was not dependent on bivalent cations. In the presence of EDTA, 0.1 mM-Mn2+ had no influence on enzyme activity, whereas 0.1 mM-Ca2+, -Co2+ and -Zn2+ decreased progesterone 5 alpha-reductase activity. Only 0.1 mM-Mg2+ was slightly stimulatory. EDTA and thiol reagents such as dithiothreitol stimulate progesterone 5 alpha-reductase activity. By means of linear sucrose gradient fractionation of the cellular membranes, progesterone 5 alpha-reductase was found to be located in the endoplasmic reticulum.

Cations↗
PubMed1991

Phospholipid requirement of progesterone 5 alpha-reductase from gastric mucosa microsomes of guinea pig.

Progesterone 5 alpha-reductase partially purified from gastric mucosa microsomes was stimulated by short-chain synthetic phosphatidylcholines (PC), such as dilauroyl PC, but not by various PC from biological sources. Phosphatidylserine (PS) activated the gastric 5 alpha-reductase to a limited extent compared to the liver 5 alpha-reductase described previously [Ichihara, K., and Tanaka, C. (1987) Biochem. Biophys. Res. Commun. 149, 482-487]. In search of more effective phospholipid activators, we tested the effects of various lysophospholipids on 5 alpha-reductase activity. Strongly stimulatory effects were observed when lysophosphatidylcholine (lysoPC) and lysophosphatidylethanolamine (lysoPE) were used instead of PC and phosphatidylethanolamine. Examination of synthetic lysoPC and lysoPE differing in acyl chain lengths showed that fatty chains of 12 to 16 carbons were effective in stimulating the 5 alpha-reductase. By contrast, other lysophospholipids such as lysophosphatidic acid, lysophosphatidylglycerol or lysophosphatidylserine (lysoPS) greatly inhibited 5 alpha-reductase activity. These findings suggest that gastric 5 alpha-reductase may be under dual regulation; lysoPC and lysoPE may play important roles as positive effectors, whereas lysophosphatidic acid, lysophosphatidylglycerol and lysoPS act as negative effectors in progesterone 5 alpha-reductase regulation.

Animals↗
PubMed1989

The kinetic mechanism of the hypothalamic progesterone 5 alpha-reductase.

The kinetic mechanism of the hypothalamic NADPH-linked progesterone 5 alpha-reductase from female rats was determined to be equilibrium ordered sequential by initial velocity, product inhibition and dead-end inhibition studies. Analysis of the initial velocity data resulted in intersecting double reciprocal plots indicating a sequential mechanism (apparent Km (progesterone) = 95.4 +/- 4.5 nM; apparent Kia(NADPH) = 9.9 +/- 0.7 microM). The plot of 1/v vs 1/progesterone intersected on the ordinate which is consistent with an equilibrium ordered mechanism. Ordered addition of the substrates was also supported by product inhibition studies with NADP versus NADPH and NADP versus progesterone. NADP is a competitive inhibitor versus NADPH (apparent Kis = 4.3 +/- 1.3 microM) and a noncompetitive inhibitor versus progesterone (apparent Kis = 31.9 +/- 1.4 microM and apparent Kii = 145.4 +/- 15.5 microM). These inhibition patterns show that NADPH binds prior to progesterone. Taken together, these analyses indicate that the cofactor, NADPH, binds to the enzyme in rapid equilibrium and preferentially precedes the binding of progesterone.

3-Hydroxysteroid Dehydrogenases↗
PubMed1984

Progesterone 5 alpha-reductase in mouse brain.

We have examined the distribution of progesterone 5 alpha-reductase activity in minces of discrete brain areas from two strains of adult ovariectomized mice known to be differentially responsive to the behavioral effects of exogenously administered 5 alpha-pregnane-3,20-dione (5 alpha-DHP). Minces of freshly dissected midbrain tegmentum (TEG), hypothalamus-preoptic area (HPOA), hippocampus (HIP), and parietal cortex (CTX) were incubated with [4-14C]progesterone in a Krebs-Henseleit bicarbonate buffer solution containing 10 mM glucose for 2 h at 37 degrees C. The [14C]5 alpha-DHP produced was identified by comparison with known standards in paper and thin-layer chromatography systems, derivative formation, and recrystallization to a constant 14C/3H ratio. We found a 6-fold difference in the extent to which various brain areas formed 5 alpha-DHP. The amount of conversion was similar for both strains of mice. When expressed as picomoles of 5 alpha-DHP formed per 2 h per 20 mg of tissue, the following order of activity was observed: TEG greater than HPOA greater than HIP greater than CTX.

3-Hydroxysteroid Dehydrogenases↗
PubMed1992

Exocyclic-keto reductase activities for progesterone and S-warfarin in hepatic microsomes from adult male rats.

Hepatic microsomes from adult male rats representing six inbred strains catalyzed quantitatively significant, NADPH dependent reductions of progesterone to the 20 beta (20R) alcohol and S-warfarin to its 11S-OH product. Microsomes from mature females and immature rats of both sexes were essentially devoid of these activities. Two strains of rat evidenced about 21% of these activities compared with the other strains and both activities were 25-81% repressed by treatment of rats with phenobarbital (PB). An excellent linear correlation was demonstrated for the two activities considering sex, age, NADPH much greater than NADH preference, PB-repression and strain differences. However, detergent latency (71%) and resistance to trypsinolysis were only observed for the keto-reductase activity with S-warfarin. Microsomes also catalyzed the reduction of progesterone to its 20 alpha-OH derivative but this activity preferred NADH greater than NADPH, was induced 2.7-fold by PB and was essentially independent of age, sex and animal strain. Furthermore, unlike the 20 beta-OH activity, this reduction was resistant to proteolytic inactivation.

20-Hydroxysteroid Dehydrogenases↗
PubMed1986

The kinetic mechanism of the anterior pituitary progesterone 5 alpha-reductase.

An analysis of the kinetic mechanism of the microsomal NADPH-linked progesterone 5 alpha-reductase obtained from female rat anterior pituitaries was performed. Initial velocity, product inhibition and dead-end inhibition studies indicate that the kinetic mechanism for the progesterone 5 alpha-reductase is equilibrium ordered sequential. Analysis of the initial velocity data resulted in intersecting double reciprocal plots suggesting a sequential mechanism [apparent Km(progesterone) = 88.2 +/- 8.2 nM; apparent Kia(NADPH) = 7.7 +/- 1.1 microM]. Furthermore, the plot of 1/v vs 1/progesterone intersected on the ordinate which is indicative of an equilibrium ordered mechanism. Additional support for ordered substrate binding was provided by the product inhibition studies with NADPH versus NADP and progesterone versus NADP. NADP is a competitive inhibitor versus NADPH (apparent Kis = 7.8 +/- 1.0 microM) and a noncompetitive inhibitor versus progesterone (apparent Kis = 9.85 +/- 2.1 microM and apparent Kii = 63.2 +/- 12.5 microM). These inhibition patterns suggest that NADPH binds prior to progesterone. In sum, these kinetic studies indicate that NADPH binds to the microsomal enzyme in rapid equilibrium and preferentially precedes the binding of progesterone.

Animals↗
PubMed1984

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.

Animals↗
PubMed1984

A high affinity inhibitor of pituitary progesterone 5 alpha-reductase.

The capacity of the 5 alpha-dihydroprogesterone analog, 4-aza-4-methyl-5 alpha-pregnane-3,20-dione (AMPD), to inhibit progesterone 5 alpha-reductase and both 5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase activities (NADPH- and NADH-linked) from the female rat anterior pituitary has been investigated. Dose response studies demonstrate that AMPD is a powerful inhibitor of pituitary progesterone 5 alpha-reduction but is ineffective at inhibiting either of the 3 alpha-hydroxysteroid oxidoreductase activities, even at concentrations up to 10 microM. A kinetic analysis of the interaction of AMPD with progesterone 5 alpha-reductase indicates that it is a competitive inhibitor vs. progesterone [Kislope = 7.2 +/- 0.6 nM; apparent Michaelis-Menten constant (Km) (progesterone) = 193 +/- 18 nM] and an uncompetitive inhibitor vs. NADPH (Kiintercept = 17.9 +/- 1.4 nM). These inhibition patterns are consistent with the view that NADPH binding precedes that of either AMPD or progesterone. Furthermore, AMPD does not appear to be an irreversible inhibitor since preincubation of the enzyme (at 37 C) with AMPD and NADPH, for periods of time up to 60 min, does not lead to a time-dependent loss of activity. The inhibition can also be readily removed by dilution, even after a 60-min preincubation with the inhibitor and NADPH. It is postulated that the selective and potent inhibition of the 5 alpha-reduction of progesterone by AMPD may be due to the steroid functioning as a transition state analog. This inhibitor should prove useful in studying the properties of progesterone 5 alpha-reductase and the function of anterior pituitary progestin metabolism.

3-Hydroxysteroid Dehydrogenases↗
PubMed1980

The effects of neurotransmitters and other cellular modulators and factors on hypothalamic and anterior pituitary delta 4-steroid (progesterone) 5 alpha-reductase activity.

A number of diverse biological compounds involved in the regulation of the hypothalamo-hypophyseal-ovarian axis have been examined for effects on the conversion of 3H-progesterone to 3H-5 alpha-dihydro-progesterone and 3H-3 alpha-hydroxy-5 alpha-pregnan-20-one by female rat hypothalamus and/or anterior pituitary. Broken cell preparations were incubated with 3H-progesterone and NADPH, and product 5 alpha-reduced progestins were quantitated by reverse isotopic dilution analysis. Progesterone 5 alpha-reductase activity was reduced up to 50% in the presence of 10(-2) to 10(-3) M serotonin in both preparations. At 10(-3) M, various indoles including n-acetylserotonin, melatonin, 5-methoxytryptamine, 5-methoxytryptophol, and 5-hydroxyindole acetic acid decreased by 10 to 30% 5 alpha-reduced product formation. At 10(-2) M, carbamylcholine and norepinephrine were without effect, while 10(-2) M dopamine reduced by 20% the 5 alpha-reduction of progesterone only in pituitary homogenates. The LHRH protease inhibitor bacitracin (2 X 10(-3) M) decreased by 10 to 40% progesterone 5 alpha-reductase activity in both tissues. By itself, LHRH did not affect the 5 alpha-reduction of progesterone nor did it potentiate the bacitracin effect. In the presence of 1 mM ATP, 100 micronM cAMP and 100 micronM cGMP increased 5 alpha-reduced product formation in the hypothalamus by 19 and 14%. The gonadotropins LH and FSH and the prostaglandins E1, E2, F1 alpha, and F2 alpha were without effect. Thus, these results and others indicate that a number of cellular components and other factors can affect the in vitro 5 alpha-reduction of progesterone in broken cell preparations.

20-alpha-Dihydroprogesterone↗
PubMed1990

The nuclear conversion of pregnenolone to progesterone and subsequent binding to the nuclear progesterone-binding protein in the guinea pig adrenal cortex: a possible regulatory role for the pregnenolone-binding protein.

Progesterone, which is normally produced in the endoplasmic reticulum, was found to be rapidly degraded in the cytosolic fraction of the guinea pig adrenal cortex in vitro. Assuming this finding reflects what happens in vivo raises a question as to the source of progesterone for interacting with a nuclear progesterone-binding protein (P4-BP) that exists in this model system. It was subsequently found that pregnenolone, which in contrast to progesterone is relatively stable in the cytosol, was converted to progesterone by endogenous nuclear 3 beta-ol dehydrogenase. It was also determined that the nuclear-derived progesterone specifically bound to the nuclear P4-BP which is distinct from the classical progesterone receptor. The guinea pig adrenocortical cytosol contains a specific pregnenolone-binding protein (P5-BP) that could be virtue of its pregnenolone binding activity regulate the conversion of pregnenolone to progesterone in the nuclear compartment and thereby reduce the binding of progesterone to the nuclear P4-BP. A partially purified P5-BP preparation markedly inhibited the nuclear conversion of pregnenolone to progesterone and reduced the binding of progesterone to the nuclear P4-BP (P5-BP did not directly inhibit binding of progesterone to the nuclear P4-BP). The ability of P5-BP to inhibit the conversion of pregnenolone to progesterone was destroyed by heat and alkaline phosphatase treatment. The binding of pregnenolone to the P5-BP, as previously reported, is regulated by phosphorylation/dephosphorylation, and alkaline phosphatase-treated P5-BP loses the ability to bind pregnenolone; this process can be reversed by a cytosolic kinase. This provides a mechanism for controlled release of bound steroid. These results suggest that P5-BP regulates the nuclear conversion of pregnenolone to progesterone and thus the binding of progesterone to the nuclear P4-BP.

Adrenal Cortex↗
PubMed2002

Transcriptional regulation of the mouse steroid 5alpha-reductase type II gene by progesterone in brain.

The steroid 5alpha-reductase (5alpha-R) plays an important physiological role in the conversion of steroid hormones such as androgen and progesterone to their 5alpha-reduced derivatives. 5alpha-R type II (5alpha-R2), one of two 5alpha-R isoforms, is thought to be a key enzyme in the generation of neuroactive steroids in the brain, particularly allopregnanolone (AP), via the production of its precursor dihydroprogesterone from progesterone. In the present study, we investigated possible regulatory mechanisms of 5alpha-R2 gene expression by steroid hormones in the female mouse brain. We first cloned mouse 5alpha-R2 (m5alpha-R2) cDNA by degenerate PCR, and found that progesterone induced 5alpha-R2 gene expression to levels detectable by in situ hybridization in female mouse brains. Functional analysis of the m5alpha-R2 gene promoter by a transient expression assay with human progesterone receptor (PR) and androgen receptor (AR) expression vectors identified a progesterone and androgen regulatory element (m5alpha-R2 PRE/ARE). Results of an electrophoretic mobility shift assay revealed that both PR and AR homodimers bound directly to m5alpha-R2 PRE/ARE sequence. These findings suggest that the gene expression of m5alpha-R2 is transcriptionally regulated by progesterone in female brains.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed2003

Anxiety levels and neurosteroid synthesis in the brains of prenatally stressed male rats.

This report presents studies of the effects of immobilization stress applied to pregnant female rats during the last third of pregnancy on anxiety levels and neurosteroid synthesis in brain structures of adult offspring. Neurosteroid synthesis was assessed in terms of changes in the activity of 5alpha-reductase, the enzyme which converts progesterone into active metabolites. Prenatal stress results in a significant decrease in the level of anxiety and an increase in movement activity among adult males. Stressed rats showed increases in progesterone-5alpha-reductase activity in the hypothalamus, hippocampus, and frontal cortex. These results provide evidence that changes in the behavior of adult male rats due to stress in the prenatal period of development may be due to the formation of active progesterone metabolites in the brain.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1990

A stereospecific enzyme of the putative biosynthetic pathway of cardenolides. Characterization of a progesterone 5 beta-reductase from leaves of Digitalis purpurea L.

Leaves of Digitalis purpurea contain an enzyme activity which catalyzes the conversion of progesterone to 5 beta-pregnane-3,20-dione. Since cardenolides without exception possess a 5 beta-configuration, 5 beta-pregnane-3,20-dione can serve as a precursor for this class of secondary metabolites. It is assumed that the enzyme is part of the putative biosynthetic pathway of cardenolides. This enzyme activity was spotted in the soluble fraction of a crude homogenate. Product formation was detected by gas chromatography and by gas chromatography/mass spectroscopy (g.c./m.s.). The enzyme had a pH optimum at 8.0 and an apparent Km value of 6 microM for progesterone. It required NADPH as a co-substrate with an apparent Km value of 22 microM. The optimum temperature in vitro was 30 degrees C. The activity was not dependent on monovalent and bivalent cations.

Cardenolides↗
PubMed2001

New progesterone esters as 5alpha-reductase inhibitors.

The pharmacological activity of four new progesterone derivatives: 4-bromo-17alpha-(p-fluorobenzoyloxy)-4-pregnene-3,20-dione (7), 4-bromo-17alpha-(p-bromobenzoyloxy)-4-pregnene-3,20-dione (8), 4-bromo-17alpha-(p-chlorobenzoyloxy)-pregnene-3,20-dione (9) and 4-bromo-17alpha-(p-toluoyloxy)-4-pregnene-3,20-dione (10) was determined. These compounds were evaluated as 5alpha-reductase inhibitors on gonadectomized hamster seminal vesicles and flank organs. The pharmacological data of this study indicate that compounds 7 and 9 having at C-17 p-fluorobenzoyloxy and p-chlorobenzoyloxy ester functions respectively showed the highest antiandrogenic effect as measured by the reduction of the weight of the seminal vesicles. In the flank organ model, the same compounds 7 and 9 exhibited a smaller diameter, 1.8 and 1.0 mm, respectively, than the commercially available finasteride 3 (2.3 mm), thus indicating a higher inhibitory effect on 5alpha-reductase enzyme. Steroid 7 showed a higher inhibitory activity on the conversion of T to DHT (Fig. 3) than the presently used finasteride, thus indicating a higher antiandrogenic effect. The nonsubstituted benzoyloxy ester (compound 15) showed a lower antiandrogenic activity as measured in the seminal vesicles model than the p-substituted benzoyloxy compounds.

5-alpha Reductase Inhibitors↗
PubMed2003

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

BACKGROUND: Recent observations indicate that human tumorous breast tissue metabolizes progesterone differently than nontumorous breast tissue. Specifically, 5alpha-reduced metabolites (5alpha-pregnanes, shown to stimulate cell proliferation and detachment) are produced at a significantly higher rate in tumorous tissue, indicating increased 5alpha-reductase (5alphaR) activity. Conversely, the activities of 3alpha-hydroxysteroid oxidoreductase (3alpha-HSO) and 20alpha-HSO enzymes appeared to be higher in normal tissues. The elevated conversion to 5alpha-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). METHODS: For 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 5alphaR type1 (SRD5A1), 5alphaR type 2 (SRD5A2), 20alpha-HSO (AKR1C1), 3alpha-HSO type 2 (AKR1C3), 3alpha-HSO type 3 (AKR1C2) and 3beta-HSO (HSD3B1/HSD3B2) in the four cell lines using 18S rRNA as an internal control. RESULTS: The relative 5alpha-reductase activity, when considered as a ratio of 5alpha-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 5alpha-pregnanes in the tumorigenic cells, respectively, than in the nontumorigenic MCF-10A cells. Conversely, the 20alpha-HSO and 3alpha-HSO activities were significantly higher (p < 0.001) in MCF-10A cells than in the other three cell types. In the MCF-10A cells, 20alpha-HSO activity was 8-14-fold higher and the 3alpha-HSO activity was 2.5-5.4-fold higher than in the other three cell types. The values of 5alphaR:20alpha-HSO ratios were 16.9-32.6-fold greater and the 5alphaR:3alpha-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 5alphaR1 (p < 0.001), and lower expression of 20alpha-HSO (p < 0.001), 3alpha-HSO2 (p < 0.001), 3alpha-HSO3 (p < 0.001) in MCF-7, MDA-MB-231 and T-47D cells than in MCF-10A cells. CONCLUSION: The findings provide the first evidence that the 5alphaR activity (leading to the conversion of progesterone to the cancer promoting 5alpha-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 5alphaR activity coincides with significantly greater expression of 5alphaR1. On the other hand, the activities of 20alpha-HSO and 3alpha-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 20alpha-HSO, 3alpha-HSO2 and 3alpha-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 5alpha-pregnanes and decreased production of anti-cancer 20alpha--and 3alpha-4-pregnenes.

20-alpha-Hydroxysteroid Dehydrogenase↗
PubMed1987

A comparison of the effects of castration and 6-methylene progesterone, a 5 alpha-reductase inhibitor, on the rat ventral prostate.

6-Methylene progesterone (6MP) is an irreversible in vitro kcat inhibitor of rat prostate 5 alpha-reductase, the enzyme which converts testosterone (T) to dihydrotestosterone (DHT). Treatment of adult rats with 6MP or diethylstilbestrol (DES) decreased the weight of the ventral prostate (VP) by 45%, while castration reduced it by 86%. Histologically, the 6MP-treated VP were indistinguishable from those of controls, while the VP from DES-treated rats showed fibrous stromal hypertrophy as in castrated rats. The prostatic hydroxyproline content, an index of collagen levels, was enhanced by castration or DES, but was not significantly increased by 6MP. Within 2 days of 6MP treatment, the 5 alpha-reductase activity was reduced by 46% and ornithine decarboxylase (ODC) activity was lowered by 27%. During this time the prostatic acid phosphatase activity increased 42% and remained elevated with continued exposure to 6MP up to 13 days. The castration-induced involution of the VP was accompanied by a reduction in serum T and an increase in serum luteinizing hormone (LH). 6MP had no effect on T and LH serum levels but reduced the DHT content within the VP by 64%. Our results indicate that the structure and secretory acid phosphatase activity of the VP are less sensitive to changes in the ratio of T:DHT than is cell proliferation. Thus, the relative amounts of DHT and T within the VP may prove to be more significant than the absolute amount of either androgen in controlling prostate growth or its attendant neoplasms.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1977

Histochemistry of 3beta-hydroxysteroid dehydrogenase in rat ovary. I. Amethodological study.

By recording the incubation time needed for initial appearance of the red and blue formazans the reliability of the histochemical method for 3beta-HSD was investigated: 1. Prefixation of small tissue blocks with 1% W/V methanol-free formaldehyde (pH=7.2) for up to 30 min preserved morphological integrity as well as maximal enzyme activity. Moreover, the substantivity of formazans and lipids was enhanced. 2. Commercial available glutaraldehyde (pH=7.2) induced SH groups in the tissue (even at 0.1% W/V for 5 min) thereby enhancing the Nothing dehydrogenase reaction. 3. Preextraction of lipids with acetone for 20 min at -30 degree C caused no loss of activity and was an inevitable step if a reliable activity pattern had to be achieved (e.g. in interstitial cells). 4. No diffusion of enzyme was noticed within 30 min of preincubation in phosphate buffer (0.2 M, pH=7.2) at 20 degree C. 5. By using the double-section incubation method no diffusion of 3beta-HSD or rediffusion of NADH or PMSH could be noticed withn 45 min of incubation, provided that low concentrations of NAD (0.1 mg/ml) and PMS (0.003 mg/ml) were balanced against the concentration of Nitro BT (0.5 mg/ml) or Tetranitro BT (1.0mg/ml). 6. The utlity of different inhibitors of alkaline phosphomonoesterase was tested and discussed. 7. By inhibiting alkaline phosphomonoesterase with 0.1 mM of L-p-bromotetramisole or 16 mM of beta-glycerophosphate, 3beta-HSD was shown to be exclusively NAD-linked. 8. Levamisole was a potent inhibitor of NADH-tetrazolium reductase as well as 3 beta-HSD, but not of NADPH-tetrazolium reductase. 9. 3beta-HSD possess SH groups requisite for the activity as this enzyme was totally inhibited by N-ethyl maleimide. 10. Whether alcohol dehydrogenases may use steroids as substrate is discussed; It is concluded that preextraction (by acetone) and/or the use of an inhibitor of alcohol dehydrogenase (1,10-phenanthroline) has to be performed. 11. Propylene glycol was a poor solvent for all substrates and was itself an excellent substrate for alcohol dehydrogenase. 12. Specifications for the ideal solvent of steroid substrates in the histochemical practice are proposed. DMSO showed to be promising as a steroid solvent (e.g. extraction of formazans was considerably lower as compared to DMF). 13. The utilization of substrates was descending in the following order (using 1 mM and 0.1 ml/ml of either DMF or DMSO): epiandrosterone, methandriol, dehydroepiandrosterone and pregnenolone. 14. If DMSO was used as solvent for pregnenolone (but not for the other substrates tested) an evident increase of activity was recorded as compared to DMF.

Alcohol Oxidoreductases↗