Specific 12β-Hydroxylation of Cinobufagin by Filamentous Fungi

Biotransformation of natural products has great potential for producing new drugs and could provide in vitro models of mammalian metabolism. Microbial transformation of the cytotoxic steroid cinobufagin was investigated. Cinobufagin could be specifically hydroxylated at the 12β-position by the fungus Alternaria alternata. Six products from a scaled-up fermentation were obtained by silica gel column chromatography and reversed-phase liquid chromatography and were identified as 12β-hydroxyl cinobufagin, 12β-hydroxyl desacetylcinobufagin, 3-oxo-12β-hydroxyl cinobufagin, 3-oxo-12β-hydroxyl desacetylcinobufagin, 12-oxo-cinobufagin, and 3-oxo-12α-hydroxyl cinobufagin. The last five products are new compounds. 12β-Hydroxylation of cinobufagin by A. alternata is a fast catalytic reaction and was complete within 8 h of growth with the substrate. This reaction was followed by dehydrogenation of the 3-hydroxyl group and then deacetylation at C-16. Hydroxylation at C-12β also was the first step in the metabolism of cinobufagin by a variety of fungal strains. In vitro cytotoxicity assays suggest that 12β-hydroxyl cinobufagin and 3-oxo-12α-hydroxyl cinobufagin exhibit somewhat decreased but still significant cytotoxic activities. The 12β-hydroxylated bufadienolides produced by microbial transformation are difficult to obtain by chemical synthesis.     

Substrate specificity for the 12beta-hydroxylation of bufadienolides by Alternaria alternata

Hydroxylation is an important route to synthesize more hydrophilic compounds of pharmaceutical significance. Microbial hydroxylation offers advantages over chemical means for its high specificity. In this study, a fungal strain Alternaria alternata AS 3.4578 was found to be able to catalyze the specific 12beta-hydroxylation of a variety of cytotoxic bufadienolides. Cinobufagin and resibufogenin could be completely metabolized by A. alternata to generate their 12beta-hydroxylated products in high yields (>90%) within 8 h of incubation. A. alternata could also convert 3-epi-desacetylcinobufagin into 3-epi-12beta-hydroxyl desacetylcinobufagin as the major product (70% yield). C-3 dehydrogenated products were detected in these reactions in fair yields, while their accumulation was relatively slow. The 12beta-hydroxylation of bufadienolides could be significantly inhibited by the substitution of 1beta-, 5-, or 16alpha-hydroxyl groups, and the 14beta,15beta-epoxy ring appeared to be a necessary structural requirement for the specificity. For the biotransformation of bufalin, a 14beta-OH bufadienolide, this reaction was not specific, and accompanied by 7beta-hydroxylation as a parallel and competing metabolic route. The biotransformation products were identified by comparison with authentic samples or tentatively characterized by high-performance liquid chromatography-diode array detection-atmospheric pressure chemical ionization-mass spectrometry analyses.     

Novel cytotoxic bufadienolides derived from bufalin by microbial hydroxylation and their structure-activity relationships

Microbial transformation was used to prepare novel cytotoxic bufadienolides. Twelve products (3-14) were obtained from bufalin (1) by the fungus Mucor spinosus. Their structures were elucidated by high-resolution mass spectroscopy (HR-MS) and extensive NMR techniques, including 1H NMR, 13C NMR, DEPT, 1H-1H correlation spectroscopy (COSY), two dimensional nuclear Overhauser effect correlation spectroscopy (NOESY), heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond coherence (HMBC). Compounds 3, 4, 9 and 11-14 are new mono- or dihydroxylated derivatives of bufalin with novel oxyfunctionalities at C-1beta, C-7beta, C-11beta, C-12beta and C-16alpha positions. The in vitro cytotoxic activities against human cancer cell lines of 3-14, together with 16 biotransformed products derived from cinobufagin (15-30) were determined by the MTT method, and their structure-activity relationships (SAR) were discussed.     

Bioconversion of 2 alpha-hydroxyprogesterone to 2-hydroxylated corticosteroids by newborn rat adrenal cells in primary culture

The bioconversion of 2 alpha-hydroxyprogesterone into 2-hydroxylated steroids was accomplished using newborn rat adrenal cells in primary culture. The products were purified using column and thin-layer chromatography, and identified by GC-MS. They resulted principally from the enzymatic reactions of 21-hydroxylation, 11 beta-hydroxylation, reduction of 20-oxo and 3-oxo groups, and epimerization of the substrate. In addition, minor metabolites resulted from 18-hydroxylation, 6 beta-hydroxylation and reduction of the 3-oxo-4-ene group. The identification of these compounds allowed us to conclude that the metabolism of 2 alpha-hydroxyprogesterone is similar to that of progesterone in this cellular system. Assuming that the 2 beta-epimers of the different metabolites arose principally from the transformation of 2 beta-hydroxyprogesterone, the specificity of the various enzyme systems seems to be similar for both epimers except in the case of the 11 beta-hydroxylation where the reaction appears stereospecific for the 2 beta-epimer. The 2 alpha-hydroxyl group on ring A seems to favor the reduction of the 3-oxo group and it does this stereospecifically to the 3 beta-structure. The epimerization of the substrate, which is most likely enzymatically induced, is the first example of steroid epimerization reported in the adrenal. This is a practical preparative method for synthesizing a variety of steroids hydroxylated at C-2 from a single substrate and could be adjusted to the production of important quantities of 2-hydroxylated metabolites of corticosteroids.     

Microbial transformation of three bufadienolides by Nocardia sp. and some insight for the cytotoxic structure-activity relationship (SAR)

Resibufogenin, cinobufagin, and bufalin are cytotoxic steroids isolated from the Chinese drug Chan'su. Biotransformation of these three bufadienolides by Nocardia sp. NRRL 5646 was investigated. Notably, resibufogenin was converted to 3-acetyl 15beta-hydroxyl bufotalin, via an unprecedented 14beta,15beta-epoxy ring cleavage and a regio-selective acetoxylation. This product showed significantly increased cytotoxic activity. The regio-selective acetylation of the 3-OH was also involved in the other reactions. The structures of metabolites were established by ESI-LC/MS and 2D NMR techniques. The in vitro cytotoxic activities against human cancer cell lines of the substrates and the transformed products were determined by the MTT method and their structure-activity relationship (SAR) was discussed. This investigation provided a useful approach to prepare new bufadienolides and the SAR research.     

Effects of deoxycholic acid and its epimers on lipid peroxidation in isolated rat hepatocytes

We studied the effects of deoxycholic acid and its three epimers with beta-hydroxyl groups (3alpha,12beta-, 3beta,12alpha-, and 3beta,12beta-dihydroxy-5beta-cholan-24-oic acids), which were hydrophilic and less cytotoxic, on lipid peroxidation to elucidate the relationship between structural features of bile acids and their effect on lipid peroxidation. Taurodeoxycholate markedly increased the production of thiobarbituric acid-reactive substances, end products of lipid peroxidation, in isolated rat hepatocytes, whereas epimers of taurodeoxycholate did not. Deoxycholic acid inhibited mitochondrial NADH dehydrogenase and NADH:ferricytochrome c oxidoreductase activities, leading to free radical generation, whereas epimers of deoxycholic acid had no effect on mitochondrial enzymes. These findings suggested that hydrophobic bile acids cause lipid peroxidation by impairment of mitochondrial function, leading to the generation of free radicals; and epimerization of alpha-hydroxyl groups in the steroid nucleus to beta-hydroxyl groups results in a decrease of the toxic effects of deoxycholic acid on lipid peroxidation.     

Inhibitory effect of 15-oxygenated sterols on cholesterol synthesis from 24,25-dihydrolanosterol

Several 15-oxygenated sterols were examined as to their inhibitory activity toward cholesterol synthesis from [24,25-3H]-24,25-dihydrolanosterol in the 10,000 X g supernatant fraction of a rat liver homogenate. At 40 microM, three 15 alpha-hydroxylated compounds, 14 alpha-ethylcholest-7-ene-3 beta,15 alpha-diol, 14 alpha-methylcholest-7-ene-3 beta,15 alpha-diol, and lanost-7-ene-3 beta,15 alpha-diol, were found to be extremely potent inhibitors (more than 90% inhibition) of dihydrolanosterol metabolism. The inhibitory effect of the C-15 substituents appeared to be in the order of: 15 alpha-hydroxyl greater than 15-ketone greater than 15 beta-hydroxyl.     

Endocrine steroid sulfotransferases: steroid alcohol sulfotransferase from human breast carcinoma cell line MCF-7

Steroid alcohol sulfotransferase (SAS) has been isolated from the cytosol of a human breast carcinoma cell line, MCF-7. This enzyme from Sephadex G-200 chromatography displayed a mol. wt of 118 KDa. The conditions for optimal enzymic activity of SAS were determined to be 20 min incubations at 45 degrees C in 0.2 M Tris buffer (pH 7.5) containing 0.06 M Mg2+. Chromatofocusing chromatography also yielded a single peak of SAS with a pI of 5.8. Results from the incubations of a series of androstane analogues revealed that SAS required a 3 beta-hydroxyl on a steroid with the trans bridge between the A and B rings. Neither the 3 beta-allylic hydroxyl group nor the A-ring phenolic 3-hydroxyl accepted the sulfate group from 3'-phosphoadenosine-5'-phosphosulfate. D-ring beta-hydroxyl groups were tolerated by the enzyme, however, alpha-hydroxyl groups on the D-ring appeared to interfere with the reaction. Sulfurylation of steroids by SAS was related inversely to the sum of the displacements of the 3-hydroxyl plus that of the 17-hydroxyl groups relative to the plane of symmetry of the dehydroepiandrosterone nucleus. This enzyme was also capable of sulfurylating short chain aliphatic alcohols, although at greatly reduced rates. 3 beta-Chloro-5-androstene-17-one and 2-nitroestradiol. 17 beta proved to be the best inhibitors of SAS.     

Estimation of ursodeoxycholic acid in human and bear biles using Clostridium absonum 7 beta-hydroxysteroid dehydrogenase

Ursodeoxycholic acid was estimated in bile samples from humans and wild North American black bears using 7 beta-hydroxysteroid dehydrogenase purified from Clostridium absonum by Procion Red affinity chromatography. The percentage ursodeoxycholic acid was calculated by two methods: (a) 7 beta-hydroxyl groups were quantified using 7 beta-hydroxysteroid dehydrogenase and 3 alpha-hydroxyl groups (total bile acids) were quantified using 3 alpha-hydroxysteroid dehydrogenase. The percentage ursodeoxycholic acid was calculated on the basis of [7 beta-hydroxyl groups]/[3 alpha-hydroxyl groups] X 100. (b) Bile was hydrolyzed with sodium hydroxide and subjected to thin-layer chromatography. Bands corresponding to cholic acid, chenodeoxycholic acid plus deoxycholic acid, and ursodeoxycholic acid were identified by the use of standards and Komarowsky's spray reagent. Total bile acids and total ursodeoxycholic acid were measured by elution of silica gel in unsprayed areas corresponding to the bile acid standards and quantification of the total bile acid in each eluate. Direct comparison of these methods validated the use of 7 beta-hydroxysteroid dehydrogenase in the estimation of ursodeoxycholic acid in the biles of black bears and of patients fed ursodeoxycholic acid for cholesterol gallstone dissolution. Relative percentages of ursodeoxycholic acid were 8-24% in four bears and 22 and 27% in the patients ingesting 500 and 750 mg ursodeoxycholic acid per day for 3 months, respectively. Predictably lower values were obtained in two control subjects and one patient ingesting 750 mg chenodeoxycholic acid per day for 3 months.     

Partial purification and properties of glucosyltransferase fromStreptomyces aureofaciens

Differential centrifugation, precipitation with ammonium sulphate and chromatography on DEAE-cellulose led to a twenty-fold purification of glucosyltransferase from Streptomyces aureofaciens B 96. The Michaelis constants for glucosyluridyl diphosphate (UDP-glucose) was 10.8 microM for 1,2-dihydroxy-9,10-anthraquinone (alizarin) 110 microM; the maximum rate of glucosylation reaction was 5.32 mumol per s per mg protein. The pH optimum was at 7.1; the flat temperature optimum was at 30 degrees C. Using some hydroxy derivatives of 9,10-anthraquinone it was found that the production of glucosides from aglycones with alpha-hydroxyl groups was about 1/8 of the values obtained with beta-hydroxyl substrates. In both types of aglycones the presence of another hydroxyl group led to a higher glucoside production.     

Bile acids. LXXIII. Synthesis of analogs of 7 alpha-hydroxy-4-cholesten-3-one as substrates for hepatic steroid 12 alpha-hydroxylase

Analogs of 7 alpha-hydroxy-4-cholesten-3-one were prepared to ascertain structural features necessary for maximal activity of hepatic microsomal 12 alpha-steroid hydroxylase. Methyl 3 alpha,7 alpha-dihydroxy-5 beta-cholane-24-carboxylate derived from chenodeoxycholic acid was oxidized at C-3 with silver carbonate/Celite. The product was hydrolyzed and dehydrogenated with SeO2 to provide 3-oxo-7 alpha-hydroxy-4-cholene-24-carboxylic acid. 5 beta-Cholestane-3 alpha,7 alpha,25-triol and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol were similarly oxidized at C-3 and dehydrogenated to provide 7 alpha,25-dihydroxy-4-cholesten-3-one and 7 alpha,12 alpha,25-trihydroxy-4-cholesten-3-one, respectively. The products were characterized by thin-layer and gas chromatography, ultraviolet, infrared, proton resonance and mass spectrometry.     

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.     

Synthesis of mono- and diglucosiduronates of metabolites of deoxycorticosterone and corticosterone and analysis by a new mass spectrometric technique

By condensing 3 alpha,21-dihydroxy-5 beta-pregnan-20-one, or its appropriate monoacetate, with methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-alpha-D-glucuronate in the Koenigs-Knorr reaction beta-D-glucosiduronates 10, 4, and 7 were obtained as polyacetate methyl esters. Alkaline hydrolysis of these substances cleaved the ester groups and gave the corresponding steroidal glucosiduronic acids 12, 6 and 8. Upon treatment with diazomethane, these acids produced the equivalent methyl esters. The C-3, the C-21 and the C-3,21 glucosiduronates of 3 alpha,21-dihydroxy-5 beta-pregnan-11,20-dione were prepared by previously reported methods and converted into the corresponding C-20 semicarbazones (14, 20 and 26). With C-20 stabilized by the semicarbazone group against reduction, it was possible to reduce the 11-oxo function in these substances to an 11 beta-hydroxyl group; after removal of the semi-carbazone moiety from these products at pH 2.0, glucosiduronic acids 18, 22 and 28 were obtained. The mass spectra of a representative group of the mono- and diglucosiduronic acids and esters were determined by utilizing fast atom bombardment and monitoring ions in both positive and negative modes of operation.     

Structural requirements for the action of steroids as quenchers of albumin fluorescence.

1. Androgens, corticoids, gestagens, estrogens and related steroids are effective quenchers of the intrinsic fluorescence of bovine serum albumin. The quenching effect involves the formation of a steroid albumin complex which formation constant (Kf) and free energy of formation (delta G 0) can be determined by fluorescence titration. The fluorimetrically determined delta G 0 values range from -6.5 to -7.5 kcal/mol. 2. 5 alpha-Androstane and 5 alpha-pregnane are effective quenchers of albumin fluorescence, in accord with the essentially hydrophobic nature of the steroid-albumin interaction. Introduction of hydroxy or oxo groups in 5 alpha-androstane decreases the fluorescence quenching action, but the effect of each group declines when other polar groups are present in the steroid molecule. Similar effects occur with 5 alpha-pregnane except that 20-hydroxy (or oxo) duo-polar derivatives are more effective than the parent hydrocarbon. 3. Comparison of delta G 0 values for steroids differing in a single grouping shows that the steroid-albumin interaction is increased by (a) the benzenoid A-ring; (b) sulfate or carboxylate ions in the vicinity of C-3; (c) the 3-oxo group in place of the 3 alpha-hydroxyl (with 5 beta-pregnane derivatives; not with 5 alpha-androstane derivatives); (d) 17 beta-acetyl or 17 beta-hydroxyethyl residues; (e) acetylated or propionated 17 beta-hydroxy groups; (f) acetylated or methylated hydroxy groups at the C-3 of estrogens; (g) delta 5 and delta 6 double bonds; and (h) the 19 beta-methyl group. The maximal variation of delta G 0 determined by affinity-enhancing groups is -0.8 kcal/mol. Conversely, the steroid-albumin interaction is decreased by introduction of (i) oxygen atoms at C-3, C-6, C-11, C-16, and C-17; (j) 17 alpha-ethynyl and 17 alpha-acetoxyl residues; (k) benzoylated or hexahydro-benzoylated beta-hydroxy groups at C-17; (l) acetylated and benzoylated hydroxy groups at C-3; and delta 1 (conjugated) double bond. Oxo groups at C-3, C-6, C-16 and the 16 alpha, 17 alpha-epoxy group are more effective than the corresponding alpha-hydroxyl in decreasing affinity, while at C-11 and C-17, the alpha-hydroxyl is more effective than the beta-hydroxyl and the oxo group. The effect of substituents is influenced by the whole molecular structure, particularly, by the stereostructure at the A/B juncture, and the presence of an oxo group at C-17. 4. The stereospecific effect of substituents at different positions in the steroid molecule suggests that with non-aromatic, A/B trans (planar) steroids, binding to albumin primarily involves the (alpha) rear surface of the B-, C- and D-ring, and possibly, the 17 beta-side chain. With estrogens and A/B cis (dihedral) steroids, the benzenoid A-ring and electron attracting groups at C-3, respectively, may participate in binding.     

Staining properties of alizarin red S for growing bone in vitro

The pH-metric titration curve of alizarin red S (ARS) showed that ARS ionizes in three stages: firstly the -SO3Na group, secondly the beta-hydroxyl group, and finally on the alkaline side the alpha-hydroxyl group. The titration of ARS solution with calcium ions indicates that complex and chelate formation between ARS and calcium are distinct. The in vitro staining experiments were carried out at different pH levels. Completely ionized ARS had the best affinity for growing bone surfaces. The next most effective staining occurred just prior to neutralization. The least effective staining by ARS was found after the neutralization point had been reached. The presence of calcium made the dye solution inactive for staining the growing bone in vitro.     

Corticosteroid side chain oxidations--II. Metabolism of 20-dihydro steroids and evidence for steroid acid formation by direct oxidation at C-21.

Rabbits have been injected with 4-14C-labelled progesterone, deoxycorticosterone and corticosterone and the corresponding 20 beta-3H-reduced steroids (20-dihydro steroids) in order to compare the influence of oxidation at C-20 on the excretion of steroid acids. Both 20 beta-reduced progesterone and deoxycorticosterone were extensively oxidized at C-20 and metabolized to 20-oxo-21-oic acids devoid of tritium. A small proportion of the acidic metabolites of [20 beta-3H]dihydro deoxycorticosterone retained tritium. By contrast the majority of the metabolites of [20 beta-3H]dihydro corticosterone were tritiated and [11 beta,20 beta-3H]-dihydroxy-4-pregnene-3-one-21-oic acid was identified as a major acidic metabolite. These results indicate that the presence of a 11 beta-hydroxyl in 20 beta-dihydro corticosterone inhibits oxidation at C-20 and provides evidence for the direct oxidation of this corticosteroid at C-21 in this species.     

Formation of biologically active oxysterols during ozonolysis of cholesterol present in lung surfactant

Exposure of the lung to concentrations of ozone found in ambient air is known to cause toxicity to the epithelial cells of the lung. Because of the chemical reactivity of ozone, it likely reacts with target molecules in pulmonary surfactant, a lipid-rich material that lines the epithelial cells in the airways. Phospholipids containing unsaturated fatty acyl groups and cholesterol would be susceptible to attack by ozone, which may lead to the formation of cytotoxic products. Whereas free radicalderived oxidized cholesterol products have been frequently studied for their cytotoxic effects, ozonized cholesterol products have not been studied, although they could reasonably play a role in the toxicity of ozone. The reaction of ozone with cholesterol yielded a complex series of products including 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al, 5-hydroperoxy-B-homo-6-oxa-cholestan-3beta,7a-diol, and 5beta,6beta-epoxycholesterol. Mass spectrometry and radioactive monitoring were used to identify the major cholesterol-derived product during the reaction of 2 ppm ozone in surfactant as 5beta,6beta-epoxycholesterol, which is only a minor product during ozonolysis of cholesterol in solution. A dose-dependent formation of 5beta,6beta-epoxycholesterol was also seen during direct exposure of intact cultured human bronchial epithelial cells (16-HBE) to ozone. Studies of the metabolism of this epoxide in lung epithelial cells yielded small amounts of the expected metabolite, cholestan-3beta,5alpha,6beta-triol, and more abundant levels of an unexpected metabolite, cholestan-6-oxo-3beta,5alpha-diol. Both 5beta,6beta-epoxycholesterol and cholestan-6-oxo-3beta,5alpha-diol were shown to be cytotoxic to cultured 16-HBE cells. A possible mechanism for cytotoxicity is the ability of these oxysterols to inhibit isoprenoid-based cholesterol biosynthesis in these cells.     

Ergosterol biosynthesis in novel melanized fungi from hypersaline environments

Halotolerant and halophilic melanized fungi were recently described in hypersaline waters. A close study of the sterol composition of such fungi, namely Hortaea werneckii, Alternaria alternata, Cladosporium sphaerospermum, Cladosporium sp., and Aureobasidium pullulans revealed the dominance of ergosterol and the presence of 29 intermediates of its biosynthesis pathway. The presence or absence of intermediates from distinct synthesis routes gave insight into the operative synthetic pathways from 4,4,14-trimethylcholesta-8,24-dien-3 beta-ol (lanosterol) to ergosterol in melanized fungi and in Saccharomyces cerevisiae, a reference yeast cultured in parallel. In all studied melanized fungi, initial methylation at C-24 took place before C-14 and C-4 demethylation, involving a different reaction sequence from that observed in S. cerevisiae. Further transformation was observed to occur through various routes. In A. alternata, isomerization at C-7 takes place prior to desaturation at C-5 and C-22, and methylene reduction at C-24. In addition to these pathways in Cladosporium spp., H. werneckii, and A. pullulans, ergosterol may also be synthesized through reduction of the C-24 methylene group before desaturation at C-5 and C-22 or vice versa. Moreover, in all studied melanized fungi except A. alternata, ergosterol biosynthesis may also proceed through C-24 methylene reduction prior to C-4 demethylation. -- Méjanelle, L., J. F. Lòpez, N. Gunde-Cimerman, and J. O. Grimalt. Ergosterol biosynthesis in novel melanized fungi from hypersaline environments. J. Lipid Res. 2001. 42: 352--358.     

Enzymic synthesis of steroid sulphates. XIV. Properties of human adrenal steroid alcohol sulphotransferase

Pure steroid alcohol sulphotransferase (EC 2.8.2.-) has the property of sulphurylating hydroxyl groups on different positions of the steroid ring. It has now been established that although only monosulphates are formed from substrates such as 3,17-diols, the position of the sulphate group depends on the relative configuration of the hydroxyl groups. Androst-5-ene-3 beta,17 beta-diol, for example, is sulphurylated mainly at the 17-position. In addition, compounds such as epitestosterone and 17 alpha-estradiol are sulphurylated at much higher rates than their respective 17 beta-epimers. It is believed that the steroid can approach the sulphurylation site via (i) ring A with the beta-side upwards, and in this mode a 3 beta-hydroxyl is sulphurylated at a higher rate than a 3 alpha-hydroxyl, or (ii) ring D with the beta-side downwards, and in this mode a 17 alpha-hydroxyl group is oriented in an analogous fashion to the 3 beta-hydroxyl in (i). The enzyme exhibits non-Michaelis-Menten kinetics within physiological concentrations (0-2 micro M) of the substrate dehydroepiandrosterone and evidence was obtained for the presence of multiple interacting steroid-binding sites. A regulatory role for the enzyme in the secretion of dehydroepiandrosterone from the human adrenal gland is proposed.