Inhibitory effect of gossypol on basal and luteinization factor-stimulated progesterone synthesis in porcine granulosa cells.

Gossypol, a polyphenolic aldehyde, inhibits steroidogenesis and the reproductive system in both sexes. The present study was undertaken to investigate whether gossypol may affect progesterone biosynthesis in cultured porcine granulosa cells isolated from small (1-2 mm) follicles (SGC). SGC were cultured with gossypol, NO donor S-nitroso-N-acetylpenicillamine (S-NAP) or the specific NO-synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME), in the presence or absence of follicular fluid isolated from large (5-8 mm) follicles (LFF) or conditioned media (CM) of granulosa cells isolated from large follicles (LGC). Gossypol enhanced the nitrite content in culture media of SGC and inhibited basal progesterone secretion by SGC. S-NAP (10(-3) M) inhibited progesterone secretion and enhanced the formation of cGMP by SGC. L-NAME had no effect on progesterone accumulation by SGC. The stimulatory effect of LFF or CM media on progesterone production by SGC in culture was also inhibited by S-NAP (10(-3)) and gossypol (10(-4) M). Moreover, gossypol inhibited forskolin-stimulated progesterone secretion, as well as substrate-enhanced conversion of 22-OH-cholesterol and pregnenolone to progesterone. These results suggest that the inhibitory effect of gossypol on progesterone secretion in culture of SGC may be mediated via NO generation.     

New thioderivatives of gossypol and gossypolone,as prodrugs of cytotoxic agents

New dithiane or dithiolane derivatives of gossypol and gossypolone were synthesized with dithiolethane or dithiolpropane in the presence of BF(3)/Et(2)O. These thioderivatives exhibited low toxicity on KB cells (human epidermoid carcinoma cells of the mouth). They react easily with electrophiles in aprotic solvents to regenerate gossypolone or to form dehydrogossypoldithianes and dehydrogossypoldithiolanes, which display higher toxicity on KB cells. In addition, the low toxicity of gossypol thioderivatives was reversed by nitric oxide donors in physiological media. These experiments suggest that gossypol and gossypolone dithianes and dithiolanes can be used as prodrugs that target tumor cells surrounded by high concentrations of nitric oxide.     

Kinetic properties of microsomal 3beta hydroxysteroid dehydrogenase-isomerase from the testis of Bufo arenarum H

3β-hydroxysteroid dehydrogenase 5-ene isomerase (3βHSD/I) activity is necessary for the biosynthesis of hormonally active steroids. A dual distribution of the enzyme was described in toad testes. The present study demonstrates that in testicular tissue of Bufo arenarum H., microsomal 3βHSD/I has more affinity for dehydroepiandrosterone (DHEA) than for pregnenolone (K_m=0.17±0.03 and 1.02 μM, respectively). The Hill coefficient for the conversion of DHEA and pregnenolone were 1.04 and 1.01, respectively. The inclusion of DHEA in the kinetic analysis of pregnenolone conversion affected V_max while K_m was not modified, suggesting a non-competitive inhibition of the conversion of pregnenolone. K_i was calculated from replot of Dixon's slope for each substrate concentration. K_i from the intercept and the slope of this replot were similar (0.276±0.01 and 0.263±0.02 μM) and higher than the K_m for DHEA. The K_m and K_i values suggest the presence of two different binding sites. When pregnenolone was present in the assays with DHEA as substrate, no effect was observed on the V_max while K_m values slightly increased with pregnenolone concentration. Consequently, pregnenolone inhibited the transformation of DHEA in a competitive fashion. These studies suggest that, in this species, the microsomal biosyntheses of androgens and progesterone are catalysed by different active sites.     

Inhibitory effects of gossypol-related compounds on growth of Aspergillus flavus

Aims: The objective of this study was to test a series of gossypol‐related compounds for growth inhibition against Aspergillus flavus. Methods and Results: A series of chiral and achiral gossypol derivatives, some natural products of the cotton plant and others prepared by synthesis from gossypol, were incorporated into agar plates to follow the rate of A. flavus isolate AF13 colony growth. All tested compounds exhibited some growth inhibition against this organism. The synthetic compounds, gossypolone and apogossypolone, exhibited greater activity than either racemic or chiral gossypol. Methylated derivatives (i.e. 6‐methoxy and 6,6′‐dimethoxy derivatives) generally exhibited less activity than the nonmethylated parent compounds. The (?)‐optical form of gossypol was found to be slightly more active than the (+)‐optical form, and this trend was observed regardless of the presence of methoxy groups at the 6‐position. Growth inhibition of gossypolone and apogossypolone was concentration dependent. For gossypolone, the 50% effective dose was 90 μg ml^?1 of medium (165 μmol l^?1). For apogossypolone, the most active compound in the study, the 50% effective dose was 19 μg ml^?1 (38·7 μmol l^?1). The presence of gossypol‐related terpenoids appeared to stimulate production of A. flavus sclerotia, although replicate variability was so large that it was not possible to determine a significant correlation between the mass of sclerotia formed and compound growth inhibition. Conclusions: The quinone derivatives of gossypol, gossypolone and apogossypolone demonstrated significant fungal growth inhibitory activity against A. flavus. Significance and Impact of the Study: These gossypol derivatives may provide a new class of fungicide for use against the mycotoxigenic fungus A. flavus.     

Estradiol and progesterone synthesis in human placenta is stimulated by calcitriol

Calcitriol exerts a diverse range of biological actions including the control of growth and cell differentiation, modulation of hormone secretion, and regulation of reproductive function. The placenta synthesizes calcitriol through the expression of CYP27B1, but little is known about local actions of this hormone in the fetoplacental unit. The objective of this study was to investigate the effects of calcitriol upon progesterone (P₄) and estradiol (E₂) secretion in trophoblasts cultured from term human placenta. Cells were incubated in the presence of calcitriol for 18 h and pregnenolone or androstenedione were subsequently added as substrates for the 3β-hydroxysteroid dehydrogenase (3β-HSD) or P450-aromatase (CYP19), respectively. Calcitriol stimulated in a dose-dependant manner E₂ and P₄ secretion. The use of a selective inhibitor of PKA prevented the effects of calcitriol upon E₂ secretion, but not on P₄. These results show that calcitriol is a physiological regulator of placental E₂ and P₄ production and suggest a novel role for calcitriol upon placental steroidogenesis.     

Dual subcellular localization of the 3β-hydroxysteroid dehydrogenase isomerase: Characterization of the mitochondrial enzyme in the bovine adrenal cortex

The enzyme 3β-hydroxysteroid dehydrogenase isomerase (3β-HSD/I) in an essential step in the biosynthesis of steroid such as progesterone, mineralo- and gluco-corticoids, estrogens and androgens in steroidogenic tissues. It is considered to be mainly localized in microsomes; however, 3β-HSD/I activity has also been described to be associated with mitochondrial preparations. In this study, we examined the subcellular distribution of 3β-HSD/I in bovine adrenocortical tissue and we characterized the catalytic properties of the enzyme present in the various cell compartments. About 30% of the total 3β-HSD/I activity was found to remain tightly associated with the purified mitochondrial pellet. The 3β-HSD/I and 3-ketoreductase activities were found in microsomes as well as in mitochondria. The 3β-HSD/I associated with the mitochondrial fraction did not required addition of exogenous NAD⁺. When the pyridine nucleotide was reduced ollowing addition of substrate of the tricarboxyllic acids cycle, the mitochondrial 3β-HSD/I activity decreased, suggesting that the enzyme utilizes NAD⁺ available from the matrix space. By contrast, the microsomal enzyme was inactive in the absence of exogenous NAD⁺. Submitochondrial fraction disclosed that 3β-HSD/I was associated (i) with the inner membrane and (ii) with a particulate fraction sedimenting in a density gradient between inner and outer membranes. This fraction was characterized as contact sites between the two membranes. 3β-HSD/I specific activity was much higher in this fraction than in the inner mitochondrial membrane. Altogether, these observations suggest that these mitochondrial intermembrane contact sites may represent a spacial organization of functional significance, facilitating both the access of cholesterol to the inner membrane where cytochrome P-450_scc is located and the rapid transformation of its product, pregnenolone, to progesterone, through 3β-HSD/I activity.     

Steroid production in toads

In Bufo arenarum, androgen biosynthesis occurs through a complete 5-ene pathway, including 5-androstane-3β,17β-diol as the immediate precursor of testosterone. Besides, steroidogenesis changes during the breeding period, turning from androgens to C₂₁-steroids such as 5-pregnan-3,20-diol, 3-hydroxy-5-pregnan-20-one and 5-pregnan-3,20-dione. In B. arenarum, steroid hormones are not involved in hCG-induced spermiation, suggesting that the steroidogenic shift to C₂₁-steroids during the breeding be not related to spermiation. The activity of 17-hydroxylase-C_17–20 lyase (CypP450_c17) decreases during the reproductive season, suggesting that this enzyme would represent a key enzyme in the regulation of seasonal changes. However, the increase in the affinity for pregnenolone of 3β-hydroxysteroid dehydrogenase (3HSD)/isomerase could also be involved. Moreover, the reduction in CypP450_c17 leading to a reduction in C₁₉-steroids, among them dehydroepiandrosterone (DHE), would contribute to the conversion of pregnenolone into progesterone, avoiding the non-competitive inhibition exerted by DHE on this transformation. Additionally, CypP450_c17 possesses a higher affinity for pregnenolone than for progesterone, explaining the predominance of the 5-ene pathway for testosterone biosynthesis. Animals in reproductive condition showed a significant reduction in circulating androgens, enhancing the physiological relevance of all the in vitro results. The in vitro effects of mGnRH and hrFSH on testicular steroidogenesis revealed that both hormones inhibited CypP450_c17 activity. In summary, these results demonstrate that, in B. arenarum, the change in testicular steroidogenesis during the reproductive period could be partially due to an FSH and GnRH-induced decrease in CypP450_c17 activity.     

Effects of temperature and medium composition on inhibitory activities of gossypol‐related compounds against aflatoxigenic fungi

Aims

To investigate the effects of temperature and medium composition on growth/aflatoxin inhibitory activities of terpenoids gossypol, gossypolone and apogossypolone against Aspergillus flavus and A. parasiticus.

Methods and Results

The compounds were tested at a concentration of 100 μg ml^?1 in a Czapek Dox (Czapek) agar medium at 25, 31 and 37°C. Increased incubation temperature marginally increased growth inhibition caused by these compounds, but reduced the aflatoxin inhibition effected by gossypol. Gossypolone and apogossypolone retained good aflatoxin inhibitory activity against A. flavus and A. parasiticus at higher incubation temperatures. However, increased temperature also significantly reduced aflatoxin production in control cultures. The effects of the terpenoids on fungal growth and aflatoxin production against the same fungi were also determined in Czapek, Czapek with a protein/amino acid addendum and yeast extract sucrose (YES) media. Growth of these fungi in the protein‐supplemented Czapek medium or in the YES medium greatly reduced the growth inhibition effects of the terpenoids. Apogossypolone displayed strong anti‐aflatoxigenic activity in the Czapek medium, but this activity was significantly reduced in the protein‐amended Czapek and YES media. Gossypol, which displayed little to no aflatoxin inhibitory activity in the Czapek medium, did yield significant anti‐aflatoxigenic activity in the YES medium.

Conclusions

Incubation temperature and media composition are important parameters involved in the regulation of aflatoxin production in A. flavus and A. parasiticus. These parameters also affect the potency of growth and aflatoxin inhibitory activities of these gossypol‐related compounds against aflatoxigenic fungi.

Significance and Impact of the Study

Studies utilizing gossypol‐related compounds as inhibitory agents of biological activities should be interpreted with caution due to compound interaction with multiple components of the test system, especially serum proteins.     

Progesterone attenuates the inhibitory effects of cardiotonic digitalis on pregnenolone production in rat luteal cells

Previous studies have shown that digoxin decreases testosterone secretion in testicular interstitial cells. However, the effect of digoxin on progesterone secretion in luteal cells is unclear. Progesterone is known as an endogenous digoxin-like hormone (EDLH). This study investigates how digitalis affected progesterone production and whether progesterone antagonized the effects of digitalis. Digoxin or digitoxin, but not ouabain, decreased the basal and human chorionic gonadotropin (hCG)-stimulated progesterone secretion as well as the activity of cytochrome P450 side chain cleavage enzyme (P450scc) in luteal cells. 8-Br-cAMP and forskolin did not affect the reduction. Neither the amount of P450scc, the amount of steroidogenic acute regulatory (StAR) protein, nor the activity of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) was affected by digoxin or digitoxin. Moreover, in testicular interstitial and luteal cells, progesterone partially attenuated the reduction of pregnenolone by digoxin or digitoxin and the progesterone antagonist, RU486, blocked this attenuation. These new findings indicated that (1) digoxin or digitoxin inhibited pregnenolone production by decreasing the activity of P450scc enzyme, but not Na(+)-K(+)-ATPase, resulting in a decrease on progesterone secretion in rat luteal cells, and (2) the inhibitory effect on pregnenolone production by digoxin or digitoxin was reversed partially by progesterone. In conclusion, digoxin or digitoxin decreased progesterone production via the inhibition of pregnenolone by decreasing P450scc activity. Progesterone, an EDLH, could antagonize the effects of digoxin or digitoxin in luteal cells.     

Extraglandular hormonal steroidogenesis in aged rats

We have examined the metabolism in vitro of [4-¹⁴C]pregnenolone by the following organs of 2.4-year-old rats: submandibular gland, stomach, duodenum, liver, lung, heart, spleen, kidney, skin, prostate, testis and adrenal. All tissues converted pregnenolone to progesterone, the highest yields being observed with adrenal, testis and skin. Androgen formation was intense in the testis and absent in the adrenal. Moreover, 17-hydroxylation of pregnenolone occurred moderately in kidney, skin and submandibular gland and markedly in duodenum and stomach, which also produced high amounts of dehydroepiandrosterone and/or 5-androstene-3β,17β-diol. Extratesticular synthesis of androstenedione and testosterone was very low. A significant formation of 20-dihydropregnenolone was observed in all tissues but stomach, duodenum and steroidogenic endocrines. Corticosteroids were not synthesized extraadrenally, except a small amount of 11-deoxycorticosterone in the testis. These results indicate that key steroid-biosynthetic enzymes, such as 3β-hydroxysteroid dehydrogenase/Δ⁵′Δ⁴ isomerase, 17β- and 20-hydroxysteroid dehydrogenases and steroid 17-monooxygenase/17,20-lyase are also expressed extraglandularly in the rat.     

Synthesis of gossypol atropisomers and derivatives and evaluation of their anti-proliferative and anti-oxidant activity

Gossypol 1, gossypolone 2, and a series of bis 3 and half Schiff's bases 4 of gossypol were synthesised and tested for anti-proliferative and anti-oxidant activity. (-)-Gossypol (-)-1 was the most potent inhibitor of the proliferation of the HPV-16 keratinocyte cell line (using an MTT viability assay) with a GI50 of 4.8 microM. The bis Schiff's base of (-)-gossypol with L-tyrosine ethyl ester (-)-3b was the most potent inhibitor of iron/ascorbate dependent lipid peroxidation (using the thiobarbituric acid test), with an IC50 of 11.7 microM, with (-)-gossypol being the next most potent of the series, with an IC50 of 13.1 microM. The results from these initial assays suggest that gossypol, as either a racemic mixture rac-1, or the individual atropisomers (-)-1 or (+)-1, has potential for the treatment of psoriasis.     

Circulating levels of pregnanolone isomers during the third trimester of human pregnancy

This study addresses the question of whether changes in the biosynthesis and metabolism of neuroactive pregnanolone isomers (PIs) might participate in the timing of parturition in humans. The time profiles of unconjugated allopregnanolone (3-hydroxy-5-pregnan-20-one, P35), pregnanolone (3-hydroxy-5β-pregnan-20-one, P35β), isopregnanolone (3β-hydroxy-5-pregnan-20-one, P3β5) and epipregnanolone (3β-hydroxy-5β-pregnan-20-one, P3β5β), pregnenolone, their polar conjugates, progesterone, 5-dihydroprogesterone (P5), and 5β-dihydroprogesterone (P5β) were monitored in the plasma of 30 healthy women during the third trimester of pregnancy, at 1-week intervals from the 30th week of gestation using GC–MS. Changes in the steroid levels were evaluated by two-way ANOVA with gestational age and subject as independent factors. The mean concentrations of free PIs ranged from 2 to 50 nmol/L, while the mean levels of their polar conjugates were 40–100× higher. The ratio of 5-PIs to progesterone significantly but inconspicuously culminated in the 35th week. The decelerating biosynthesis of free 5β-PIs from the 31st week and their escalating sulfation was found from the 30th week. The changes were particularly evident in the second most abundant PI pregnanolone that may, like the allopregnanolone, sustain the pregnancy via attenuation of hypothalamic GABA_A-receptors and prevent uterine contractility via binding to nuclear pregnane X receptor.     

Follicular fluid stimulation of 3 beta-hydroxysteroid dehydrogenase activity in vitro

Follicular fluid from porcine antral follicles stimulates progesterone secretion by porcine granulosa cells from small antral follicles in vitro. Fluid from large (6-12 mm) follicles has more stimulatory activity than fluid from smaller follicles. In this study, we have examined the action of charcoal-treated and filtered follicular fluid on 3 beta-hydroxysteroid dehydrogenase activity (3 beta-HSD) and the ability of exogenous pregnenolone to increase progesterone secretion. Granulosa cells cultured with 30% follicular fluid in TC 199 (v/v) for 3 days were less dependent on the presence of exogenous pregnenolone to enhance their progesterone secretion and exhibited more 3 beta-HSD activity than control cells incubated in 30% serum in TC 199. The apparent Vmax of 3 beta-HSD was increased 80% in follicular fluid-treated cells over that observed in controls (4.8 vs. 2.6 nM/min/100 mg protein) whereas the apparent Kms for 3 beta-HSD were similar (1.3 +/- 0.34 microM) in both experimental and control cells.     

Progesterone blockade of the positive feedback effects of 17β oestradiol in the ewe

Ovariectomized ewes (n = 24) were treated with implants that resulted in circulating concentrations of progesterone and 17β-oestradiol similar to those seen in intact ewes in the luteal phase of an oestrous cycle. Progesterone implants were left in for 10 days, and 17β-oestradiol implants for 14 days. Twelve of these ewes received daily injections of 17β-oestradiol in oil (i.m.) at doses sufficient to cause a surge release of luteinizing hormone (LH) in the absence of progesterone. The other 12 ewes were treated daily with vehicle (oil). Following progesterone withdrawal on Day 10, each group of 12 ewes was divided into three subgroups. Ewes in each subgroup of the groups treated daily with 17β-oestradiol or vehicle, received an injection of either 17β-oestradiol (oil i.m.), gonadotrophin-releasing hormone (GnRH) (saline, i.v.) or vehicle, 24 h after progesterone withdrawal. Following progesterone withdrawal, no LH surge was detected in ewes treated with vehicle. Surge secretion of LH was detected in ewes of all other groups. The data suggested that in progesterone-treated ewes, daily exposure to stimulatory doses of 17β-oestradiol did not desensitize the hypothalamic pituitary axis to the positive feedback effects of 17β-oestradiol. Daily exposure to 17β-oestradiol did not suppress pituitary responsiveness to GnRH. It was concluded that circulating concentrations of progesterone, similar to those seen during the luteal phase of an oestrous cycle in intact ewes, may prevent all necessary components of the LH surge secretory mechanism from responding to 17β-oestradiol.     

Purification and characterization of 3β-hydroxysteroid-dehydrogenase/isomerase from bovine adrenal cortex

The formation of 4-ene-3-ketosteroids from 3β-hydroxy-5-ene precursors is an obligatory step in the biosynthesis of hormonal steroids such as glucocorticoids, mineralocorticoids, estrogens and androgens. In the adrenal cortex, pregnenolone, 17-hydroxy-pregnenolone and dehydroisoandrosterone are converted to progesterone, 17-hydroxy-progesterone and androstenedione, respectively, by the enzymatic system 3β-hydroxy-5-ene steroid dehydrogenase and 3-keto-5-ene steroid isomerase (3β-HSD/I).

The present work reports a two step purification procedure which yields an homogenous preparation of 3β-HSD/I from bovine adrenal cortex. It uses solubilization of the microsomal proteins followed by two chromatographic steps, i.e. DEAE-cellulose and heparine-sepharose columns. The enzyme was obtained as an homogeneous protein exhibiting an apparent molecular size of 45 kDa upon SDS-gel electrophoresis and of 81 kDa upon gel filtration. The purified enzyme exhibits both the 5-ene-3β-ol steroid dehydrogenase and isomerase activities in contrast to previous work using a more complex procedure which yielded a final preparation having lost its isomerase activity [Hiwatashi et al., Biochem. J. 98 (1985) 1519–1525]. N-terminal aminoacid (29 residues) sequence of the purified protein was determined and was found identical to that predicted from the nucleic acid sequence of the recently identified enzyme cDNA [Zhas et al. FEBS Lett. 259 (1989) 153–157].     

Increasing progesterone secretion and 3β-hydroxysteroid dehydrogenase activity of human cumulus cells and granulosa-lutein cells concurrent with successful fertilization of the corresponding oocyte

In many studies it has been documented that the induction of multiple follicular growth in humans results in an asynchrony between the degree of cumulus mucification, oocyte meiotic maturation, fertilizability, and follicular cell progesterone (P₄) secretion. The present study was carried out on oocytes enclosed in fully mucified cumulus. Thus, oocyte fertilizability was correlated to human cumulus cell (hCC) and human granulosa-lutein (G-L) cell competence for P₄ secretion in culture. In the G-L cells, P₄ secretion and percentage of cells manifesting 3β-hydroxysteroid dehydrogenase (3β-HSD) activity increased concurrently with the period of culture. In the hCC, however, P₄ secretion decreased concurrently with elongation of the culture period, whereas the percentage of 3β-HSD-positive cells increased. In hCC corresponding to the fertilized oocytes, P₄ accumulation in culture medium was 1.9-fold (P < 0.001) and 1.6-fold (P < 0.02) higher on days 0–3 and 3–5 of culture, respectively, as compared to P₄ accumulation in hCC of unfertilized oocytes. Also, in hCC corresponding to the fertilized oocytes, the degree of 3β-HSD activity was found to be significantly higher shortly after aspiration and after either 3 or 5 days, compared to hCC of unfertilized oocytes. In the G-L cells pooled from all follicles yielding mature cumulus-oocyte complexes, P₄ accumulation and percentage of 3β-HSD-positive cells increased concurrently with the increase in percentage of fertilized eggs of each individual woman. These results indicate that in stimulated cycles, follicles yielding mature cumulus-oocyte complex, oocyte fertilizability, and G-L cell or hCC competence for P₄ secretion are correlated and synchronous.     

Control of bovine placental progesterone synthesis: roles of cholesterol availability and calcium-activated systems

It was previously reported that dispersed bovine placentome secretes progesterone and that the steroidogenic activity of these cells is stimulated by a calcium-mediated, cyclic nucleotide independent mechanism. In the present study, the influence of substrate availability was explored and the roles of calmodulin and protein kinase C in progestin production examined. Incubation of dispersed fetal cotyledon cells with 25-hydroxycholesterol (25-OH-C), a soluble sterol which readily enters cells and is metabolized to steroid hormones, increased progesterone secretion in a dose-dependent manner. The response to 25-OH-C was dependent on the extracellular calcium concentration. Methyl isobutyl xanthine (MIX) alone also increased pregnenolone as well as progesterone secretion, and the combination of 25-OH-C and MIX stimulated progesterone secretion was inhibited by trifluoperazine. The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), caused no major effects on steroidogenesis but the stimulatory effects of MIX or the ionophore A23187 were enhanced in its presence. These findings suggest that (1) basal progesterone secretion by fetal cotyledon cells is limited by cholesterol availability; (2) MIX increases steroidogenesis in part by increasing the synthesis of pregnenolone, but its actions are expressed independently of cholesterol availability; (3) both calmodulin and protein kinase C may participate in the modulation of bovine placental steroidogenesis.     

Steroid hydroxylations by guinea-pig adrenal tissue fractions

The effect of oxidizable substrates, -ketoglutarate and isocitrate on 11β-hydroxylation in guinea-pig adrenal mitochondria has been studied. These substrates supported the conversion of Compound S (17,21-dihydroxy-pregnene-3,20-dione) into cortisol as well as exogenously added NADPH in Ca²⁺-swollen mitochondria. Progesterone was also hydroxylated at the C-11β position but not at the C-21 position. Microsomes, on the other hand, when NADPH was added, converted pregnenolone, progesterone and 17-hydroxyprogesterone into Compound S, but showed no steroid 11β-hydroxylating activity. Evidence obtained in incubations carried out with -ketoglutarate and isocitrate in the presence of 2,4-dinitrophenol, oligomycin, amytal and antimycin A indicate that -ketoglutarate utilization for steroid 11β-hydroxylation is dependent on activity of the classical electron chain. This activity can be related to high energy intermediates possibly needed for NADPH reduction arising from NADH oxidation via the energy-requiring transhydrogenase reaction. These reactions do not appear necessary for isocitrate utilization and isocitrate oxidation probably gives rise to intramitochondrial NADPH reduction as a result of a NADP⁺-linked isocitrate dehydrogenase. Data obtained in oxygen uptake studies with an antimycin A blocked system supplied with -ketoglutarate, are in accordance with this conclusion. The high-speed supernatant fraction (103000 × g) could partially replace -ketoglutarate, isocitrate or Ca²⁺ + NADPH, indicating that it contains a factor(s) (the physiological substrate?) which brings about intramitochondrial NADPH.     

Studies on Baeyer–Villiger oxidation of steroids: DHEA and pregnenolone d-lactonization pathways in Penicillium camemberti AM83

Penicillium camemberti AM83 strain is able to carry out effective Baeyer–Villiger type oxidation of DHEA, pregnenolone, androstenedione and progesterone to testololactone. Pregnenolone and DHEA underwent oxidation to testololactone via two routes: through 4-en-3-ketones (progesterone and/or androstenedione respectively) or through 3β-hydroxy-17a-oxa-d-homo-androst-5-en-17-one.Analysis of transformation progress of studied substrates as function of time indicates that the 17β-side chain cleavage and oxidation of 17-ketones to d-lactones are catalyzed by two different, substrate-induced, BVMOs. In the presence of a C-21 substrate (pregnenolone or progesterone) induction of the enzyme catalyzing cleavage at 17β-acetyl chain was observed, whereas DHEA and androstenedione induced activity of the BVMO responsible for the ring-D oxidation; 5-en-3β-alcohol was a more effective inducer that the respective 4-en-3-ketone.