Modifications of Δ5-3-ketosteroid isomerase induced by ultraviolet irradiation in the presence of the solid-phase photoaffinity reagent Δ6-testosterone agarose

The nature of the products formed during the photoinactivation of Δ⁵-3-ketosteroid isomerase in the presence of the solid-phase photoaffinity reagent Δ⁶-testosterone succinyl agarose has been investigated after ultraviolet irradiation. The polypeptide products eluted from the agarose phase by sodium cholate, sodium dodecyl sulfate, and pH 10.5 triethylamine buffer have been characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis, pH 4–6 gel isoelectric focusing, and amino acid analysis. The amino acid compositions of the cholate eluted and SDS eluted products are found to be similar to that of native isomerase, whereas the covalently bound polypeptide eluted by pH 10.5 triethylamine possesses a distinetly different composition. Digestion of the covalently bonded isomerase polypeptide with trypsin yields an agarose-bound peptide fraction that has been characterized by its amino acid composition. This composition is different from that of the undigested covalently bound polypeptide and suggests that the site of covalent attachment lies somewhere between residues 28 and 45 of the isomerase polypeptide.     

Solubilization and separation of delta5,3beta-hydroxysteroid dehydrogenase and 3-oxosteroid-delta4-delta5-isomerase from bovine adrenal cortex microsomes.

A physical separation of delta5,3beta-hydroxysteroid dehydrogenase and 3-oxosteroid delta4-delta5-isomerase solubilized from bovine adrenocortical microsomes is described for the first time. The solubilization as well as the separation was carried out with a mixture of a detergent: a substituted betaine (Empigen BB/P) and sodium cholate. This latter detergent protects isomerase from complete inactivation by Empigen and is necessary for the recovery of a significant amount of soluble isomerase. Separation of dehydrogenase and isomerase was successfully accomplished by the use of a DEAE-Biogel A anion-exchanger. Dehydrogenase activity was eluted, while the isomerase was retained. Measurements of dehydrogenase activity with androst-5-en-3beta-ol-17-one, pregnen-3beta-ol-20-one and pregn-5-en-(3beta,17alpha)-diol-20-one and of isomerase activity with androst-5-en-(3,17)-dione and pregn-5-en-(3,20)-dione suggested that more than one isomerase and more than one dehydrogenase form were present.     

Use of a solid-phase photoaffinity reagent to label a steroid binding site: application to the delta 5-3-ketosteroid isomerase of Pseudomonas testosteroni

In order to extend our analysis of the reactions that occur during the active site directed photoinactivation of delta 5-3-ketosteroid isomerase sensitized by unsaturated steroid ketone photoaffinity reagents, the site of covalent attachment has been identified. A solid-phase photoaffinity reagent, delta 6-testosterone-agarose, has been employed for this purpose; this type of reagent, in contrast to solution-phase reagents, facilitated the recovery of a peptide fragment of the isomerase bearing the residue at which covalent attachment had occurred. Amino acid analysis and sequence determination of the peptide provided evidence that the site of attachment was aspartate-38. This result, in combination with the low-resolution crystallographic structure of the enzyme [Westbrook, E. M., Piro, O. E., & Sigler, P. B. (1984) J. Biol. Chem. 259, 9096-9103], suggests that aspartate-38 is located in the vicinity of the bottom of the steroid-binding pit. The potential usefulness of solid-phase photoaffinity reagents in the identification of sites of covalent attachment on target proteins such as hormone receptors is discussed.     

Photoaffinity labeling of the oxysterol receptor

A cytosolic receptor protein for oxygenated sterols, postulated to be involved in the regulation of 3-hydroxy-3-methylglutaryl-CoA reductase and cholesterol biosynthesis, can be labeled covalently by photoactivation of 7,7'-azo-[5,6-3H]cholestane-3 beta,25-diol. Other compounds tested for their potential as photoaffinity reagents were: 25-hydroxycholesta-4,6-dien-3-one, 3 beta,25-dihydroxycholest-5-en-7-one, and 3 beta-hydroxycholesta-8(14),9(11)-dien-15-one. These sterols did not bind to the receptor with adequate affinity, were not readily photolyzed, or did not react covalently with the receptor during photolysis. The successful photoaffinity label, 7,7'-azocholestane-3 beta,25-diol, binds to the receptor with high affinity (Kd = 9.1 nM). After activation of the partially purified oxysterol-receptor complex with UV light (greater than 300 nm), several covalently labeled proteins were found upon sodium dodecyl sulfate-gel electrophoresis. Labeling of one protein, Mr approximately 98,000, was much reduced when the binding reaction was carried out in the presence of an excess of unlabeled oxysterol. Under the reaction conditions investigated so far, approximately 1% of the specifically bound sterol was covalently linked after photolysis. These results are consistent with previous information suggesting that the Mr of the receptor subunit is approximately 97,000. The covalent labeling of the receptor reported herein should facilitate its further purification and characterization.     

Preparation of 17 alpha-iodoethynylandrosta- and 17 alpha-(2-iodoethenyl)androsta-4,6-dien-17 beta-ol-3-ones as active site-directed photoaffinity ligands for androgen-binding proteins.

Unsaturated analogues of androst-4-en-17 beta-ol-3-one, each with a 17 alpha-iodoethynyl or 17 alpha-(2-iodoethenyl) substituent, were prepared, and their relative binding affinities (RBAs) for androgen-binding protein (ABP) were compared with those of 5 alpha-androstan-17 beta-ol-3-one, androst-4-en-17 beta-ol-3-one, androsta-4,6-dien-17 beta-ol-3-one, and androsta-1,4,6-trien-17 beta-ol-3-one. These binding studies indicate that the iodine[125I] analogues of 17 alpha-iodoethynyl and 17 alpha-[(E)-2-iodoethenyl] derivatives of androsta-4,6-dien-17 beta-ol-3-one and androsta-1,4,6-trien-17 beta-ol-3-one will have RBAs at least twice as great as that of 5 alpha-androstan-17 beta-ol-3-one. They can be prepared from 17 alpha-ethynylandrosta-4-en-17 beta-ol-3-one, the final synthetic step using N-[125I]iodosuccinimide, and are potential radioiodinated, active site-directed photoaffinity ligands for ABP and testosterone-binding globulin.     

The degradation of cholic acid by Pseudomonas sp. N.C.I.B. 10590 under anaerobic conditions.

The bacterial degradation of cholic acid under anaerobic conditions by Pseudomonas sp. N.C.I.B. 10590 was studied. The major unsaturated neutral compound was identified as 12 beta-hydroxyandrosta-4,6-diene-3,17-dione, and the major unsaturated acidic metabolite was identified as 12 alpha-hydroxy-3-oxochola-4,6-dien-24-oic acid. Eight minor unsaturated metabolites were isolated and evidence is given for the following structures: 12 alpha-hydroxyandrosta-4,6-diene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-4,6-dien-3-one, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-1,4,6-trien-3-one, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-1,4,6-trien-3-one, 12 alpha-hydroxyandrosta-1,4-diene-3,17-dione, 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione, 3,12-dioxochola-4,6-dien-24-oic acid and 12 alpha-hydroxy-3-oxopregna-4,6-diene-20-carboxylic acid. In addition, a major saturated neutral compound was isolated and identified as 3 beta,12 beta-dihydroxy-5 beta-androstan-17-one, and the only saturated acidic metabolite was 7 alpha,12 alpha-dihydroxy-3-oxo-5 beta-cholan-24-oic acid. Nine minor saturated neutral compounds were also isolated, and evidence is presented for the following structures: 12 beta-hydroxy-5 beta-androstane-3,17-dione, 12 alpha-hydroxy-5 beta-androstane-3,17-dione, 3 beta,12 alpha-dihydroxy-5 beta-androstan-17-one, 3 alpha,12 beta-androstan-17-one, 3 alpha,12 alpha-dihydroxy-5 beta-androstan-17-one, 5 beta-androstane-3 beta,12 beta,17 beta-triol, 5 beta-androstane-3 beta,12 alpha,17 beta-triol, 5 beta-androstane-3 alpha,12 beta,17 beta-triol and 5 beta-androstane-3 alpha,12 alpha,17 beta-triol. The induction of 7 alpha-dehydroxylase and 12 alpha-dehydroxylase enzymes is discussed, together with the significance of dehydrogenation and ring fission under anaerobic conditions.     

Comparison of rabbit androgen binding protein with testosterone estradiol binding globulin--I. Physical and chemical properties

Rabbit epididymal androgen binding protein (rbABP) and serum testosterone estradiol binding globulin (rbTeBG) were purified and their physicochemical properties compared. Both proteins bound dihydrotestosterone (DHT) with high affinity. Both contained two components, Heavy (H) and Light (L), and their molecular weights and pI values were comparable. rbABP and rbTeBG were different with regard to their ConA-Sepharose binding property. rbABP was not bound by ConA-Sepharose while rbTeBG was found and retained by this lectin; thus, rbABP and rbTeBG differed in their carbohydrate structure. Peptide mapping on SDS-PAGE indicated that the H components of rbABP and rbTeBG were distinct even though they showed a high degree of homology. By contrast, the L components of these two proteins appeared to be identical. The structure of the steroid binding sites of these two proteins was analyzed by peptide mapping of [1,2(3)H]17 beta hydroxy-androsta-4,6-dien-3-one photoaffinity labeled protein. The size distribution of radioactive peptide fragments generated appeared to be identical for these two proteins. However, the distribution of labeled peptides was slightly different when examined by high pressure liquid chromatography (HPLC). The observations suggest that the differences between rbABP and rbTeBG might reside not only in carbohydrate moieties but also in their amino acid sequences.     

Transformation of 5-ene steroids by the fungus Aspergillus tamarii KITA: Mixed molecular fate in lactonization and hydroxylation pathways with identification of a putative 3β-hydroxy-steroid dehydrogenase/Δ–Δ isomerase pathway

The fungus Aspergillus tamarii metabolizes progesterone to testololactone in high yield through a sequential four step enzymatic pathway which, has demonstrated flexibility in handling a range of steroidal probes. These substrates have revealed that subtle changes in the molecular structure of the steroid lead to significant changes in route of metabolism. It was therefore of interest to determine the metabolism of a range of 5-ene containing steroidal substrates. Remarkably the primary route of 5-ene steroid metabolism involved a 3β-hydroxy-steroid dehydrogenase/Δ⁵–Δ⁴ isomerase (3β-HSD/isomerase) enzyme(s), generating 3-one-4-ene functionality and identified for the first time in a fungus with the ability to handle both dehydroepiansdrosterone (DHEA) as well as C-17 side-chain containing compounds such as pregnenolone and 3β-hydroxy-16α,17α-epoxypregn-5-en-20-one. Uniquely in all the steroids tested, 3β-HSD/isomerase activity only occurred following lactonization of the steroidal ring-D. Presence of C-7 allylic hydroxylation, in either epimeric form, inhibited 3β-HSD/isomerase activity and of the substrates tested, was only observed with DHEA and its 13α-methyl analogue. In contrast to previous studies of fungi with 3β-HSD/isomerase activity DHEA could also enter a minor hydroxylation pathway. Pregnenolone and 3β-hydroxy-16α,17α-epoxypregn-5-en-20-one were metabolized solely through the putative 3β-HSD/isomerase pathway, indicating that a 17β-methyl ketone functionality inhibits allylic oxidation at C-7. The presence of the 3β-HSD/isomerase in A. tamarii and the transformation results obtained in this study highlight an important potential role that fungi may have in the generation of environmental androgens.     

Isolation of rabbit muscle glucosephosphate isomerase by a single-step substrate elution.

A simple method has been developed for the rapid isolation of crystalline glucosephosphate isomerase (EC 5.3.1.9) from rabbit muscle. The enzyme is first bound to cellulose phosphate by adding the ion exchanger to a solution of the crude tissue extract. After filtering and washing the cellulose with buffer, the isomerase is specifically eluted in a batch process by its substrate, glucose 6-phosphate. The entire procedure is very rapid and results in a good recovery (at least 50%) of the enzyme with specific activity of approximately 900 units per mg. The enzyme is homogeneous by polyacrylamide gel electrophoresis in the presence of absence of sodium dodecyl sulfate and by analytical ultracentrifugation.     

Detection of a transient enzyme-steroid complex during active-site-directed irreversible inhibition of 3-oxo-delta 5-steroid isomerase

The reaction of the active-site-directed irreversible inhibitor (17S)-spiro[estra-1,3,5(10),6,8-pentaene-17,2'-oxiran]-3-ol (5 beta) with 3-oxo-delta 5-steroid isomerase has been monitored by repetitive scanning ultraviolet spectroscopy of a solution of 5 beta plus isomerase against a blank containing only 5 beta. Upon initial mixing of 5 beta with the isomerase an absorbance maximum at ca. 250 nm appears. With time, this peak decreases and is replaced with a new peak near 280 nm. These results directly demonstrate the existence of a transient enzyme-steroid intermediate in the inactivation reaction. The ultraviolet spectrum suggests that the steroid in the transient complex resembles the ionized phenol, while the phenolic group in the irreversibly bound complex is un-ionized. These spectral studies support our previous proposal that there are two enzyme-steroid complexes that are related by a 180 degree rotation about an axis perpendicular to the plane of the steroid nucleus. This hypothesis offers an explanation for the reaction of 17 beta-oxiranes with the same residue (Asp-38) that is thought to be involved in the catalytic mechanism. Two new oxiranes, (17S)-spiro[estra-1,3,5(10)-triene-17,2'-oxiran]-3 beta-ol (6 beta) and (17S)-spiro[5 alpha-androstane-17,2'-oxiran]-3-one (8 beta), were also found to be potent active-site-directed irreversible inhibitors of the isomerase (k3/KI = 31 M-1 s-1 and 340 M-1 s-1, respectively). The relationship of these results to the nature of the active site of the isomerase is discussed.     

Studies on the interaction of matrix-bound inhibitor with Band 3, the anion transport protein of human erythrocyte membranes

The effect of temperature and chemical modification on the interaction of the human erythrocyte Band 3 protein (the anion transport protein) with 4-acetamido-4'-isothiocyanostilbene 2,2'-disulfonate (SITS; Ki = 10 microM)-Affi-Gel 102 resin was studied. Band 3 binds to the affinity resin in two states; weakly bound, which is eluted by 1 mM 4-benzamido-4'-aminostilbene 2,2'-disulfonate (BADS; Ki = 2 microM), and strongly bound, which is eluted only under denaturing conditions by 1% lithium dodecyl sulfate (LDS). At 4 degrees C, most of band 3 was present initially in the weakly bound form and very little in the strongly bound form. With longer incubations at 4 degrees C, the weakly bound form was slowly converted to the strongly bound form. At 37 degrees C, most of Band 3 was rapidly converted to the strongly bound form, with some Band 3 still remaining in the weakly bound form. Band 3 dimers, labelled with 4,4'-diisothiocyanostilbene 2,2'-disulfonate (DIDS) in one monomer, did bind to immobilized SITS but did not become tightly bound upon incubation at 37 degrees C. Since the covalent attachment of DIDS to one monomer prevented the adjacent monomer from becoming tightly bound to immobilized SITS ligand, this observation suggests that the inhibitor-binding sites of the two adjacent monomers must be interacting with each other. When the inhibitor site of Band 3 was selectively modified by citrate in the presence of 1-ethyl-3-(3-azonia-4,4-dimethylpentyl)carbodiimide (EAC), Band 3 bound to the resin was more easily eluted by BADS, suggesting reduced affinity for immobilized SITS. However, citrate-modified Band 3 did become tightly bound upon incubation at 37 degrees C.     

Antiprogestational agents. The synthesis of 7-alkyl steroidal ketones with anti-implantational and antidecidual activity

A series of 7α- and 7β- alkyl derivatives of steroidal 4-en- and 5-en-3-ones were prepared by 1,6-conjugate addition of organocopper reagents to various steroidal 4,6-dien-3-ones of the androstane, estrane and gonane series. Biological study of these and related compounds revealed that 17β-hydroxy-7α-methyl-5-androsten-3-one (2), 17β-hydroxy-7α-methyl-5-estren-3-one acetate and 17β-hydroxy-7α-methyl-4-estren-3-one acetate had significant anti-implantational and antidecidual activities. The contragestative effects were associated with the latter antihormonal properties, and not with the androgenicity of these compounds.     

Synthesis of 3α,7α-dihydroxy-5β-androstan-17-one

A short and efficient method for the stereospecific synthesis of 3α,7α-dihydroxy-5β-androstan-17-one was accomplished from the readily available 4-androstene-3,17-dione. Key steps are the stereospecific and selective epoxidation of 4,6-androstadiene-3,17-dione, followed by hydrogenations with carefully selected reagents, solvents and reaction conditions.     

Isolation of histidyl peptides of the steroid-binding site of human placental estradiol 17β-dehydrogenase

Human placental estradiol 17β-dehydrogenase (E.C. 1.1.1.62) was inactivated at pH 6.3 by 3-bromo[2′-¹⁴C]acetoxy-1,3,5(10)estratrien-17-one, a known substrate. The affinity-alkylated enzyme was then hydrolyzed by trypsin. Radioactive peptides were initially isolated by gel filtration and identified according to which residue was alkylated. Tryptic peptides containing radioactive 3-carboxymethylhistidyl residues were further purified by cation-exchange chromatography. The population of these peptides varied, depending upon the conditions of enzyme inactivation. With 60 μM 3-bromo[2′-¹⁴C]acetoxy-1,3,5(10)estratrien-17-one four major peptides (a,b,c,d) each containing radioactive 3-carboxymethylhistidine, were eluted from the cation-exchange column. The alkylation of all of these peptides was completely suppressed when the enzyme was inactivated in the presence of excess estradiol-17β. The presence of equimolar NADPH during incubation greatly enhanced the alkylation of all four peptides. In the presence of NADPH, estradiol-17β most significantly decreased the formation of peptide d. Peptide d was the only peptide identified when the concentration of the alkylating steroid was lowered to 6 βM, a value approaching the Km. These observations indicate that peptide d is a histidyl-bearing peptide from the steroid-binding site which proximates the steroid A-ring. They further suggest that with the affinity labeling steroid at higher concentrations other nonspecific, hydrophobic sites on the enzyme are occupied and labeled.     

Stereoselective reduction of C-2 substituted steroid C-3 ketones with lithium tris-(R,S-1,2-dimethylpropyl)-borohydride and sodium borohydride

The effect of C-2 substitution on the stereoselective reduction of steroid C-3 ketones with lithium tris-(R,S-1,2-dimethylpropyl)-borohydride and sodium borohydride was investigated. The C-2 mono- and di-substituted chloro and methyl derivatives were predominantly reduced to one of the epimeric alcohols. The 2 alpha-chloro and 2 alpha-methyl derivatives of 17 beta-acetoxy-5 alpha-androstan-3-one undergo stereoselective reduction with lithium tris-(R,S-1,2-dimethylpropyl)-borohydride to the axial (3 alpha) alcohol as observed in the unsubstituted compound, whereas sodium borohydride gives predominantly the equatorial (3 beta) alcohol. The 2 beta-chloro, 2 beta-methyl, 2,2-dichloro, and 2,2-dimethyl derivatives are reduced predominantly to the equatorial (3 beta) alcohol by both reagents.     

Inactivation of delta 5-3-oxo steroid isomerase with active-site-directed acetylenic steroids.

Several steroid analogues containing conjugated acetylenic ketone groups as part of a seco-ring structure or as substituents on the intact steroid system are irreversible inhibitors of delta 5-3-oxo steroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni. Thus 10 beta-(1-oxoprop-2-ynyl)oestr-4-ene-3,17-dione (I), 5,10-seco-oestr-4-yne-3,10,17-trione (II), 17 beta-hydroxy-5,10-seco-oestr-4-yne-3,10-dione (III) and 17 beta-(1-oxoprop-2-ynyl)androst-4-en-3-one (IV) irreversibly inactivate isomerase in a time-dependent manner. In all cases saturation kinetics are observed. Protection against inactivation is afforded by the powerful competitive inhibitor 19-nortestosterone. The inhibition constants (Ki) for 19-nortestosterone obtained from such experiments are in good agreement with those determined from conventional competitive-inhibition studies of enzyme activity. These compounds thus appear to be active-site directed. In every case the inactivated enzyme could be dialysed without return of activity, indicating that a stable covalent bond probably had formed between the steroid and enzyme. Compound (I) is a very potent inhibitor of isomerase [Ki = 66.0 microM and k+2 = 12.5 x 10(-3) s-1 (where Ki is the dissociation constant of the reversible enzyme-inhibitor complex and k+2 is the rate constant for the inactivation reaction of the enzyme-inhibitor complex)] giving half-lives of inactivation of 30-45 s at saturation. It is argued that the basic-amino-acid residue that abstracts the intramolecularly transferred 4 beta-proton in the reaction mechanism could form a Michael-addition product with compound (I). In contrast, although compound (IV) has a lower inhibition constant (Ki = 14.5 microM), it is a relatively poor alkylating agent (k+2 = 0.13 x 10(-3) s-1). If the conjugated acetylenic ketone groups are replaced by alpha-hydroxyacetylene groups, the resultant analogues of steroids (I)-(IV) are reversible competitive inhibitors with Ki values in the range 27-350 microM. The enzyme binds steroids in the C19 series with functionalized acetylenic substituents at C-17 in preference to steroids in the C18 series bearing similar groups in the ring structure or as C-10 substituents. In the 5,10-seco-steroid series the presence of hydroxy groups at both C-3 and C-17 is deleterious to binding by the enzyme.     

Chemoselective reduction of 1,4,6-cholestatrien-3-one and 1,4,6-androstatriene-3,17-dione by various hydride reagents

The chemoselectivity of rigid cyclic alpha,beta-unsaturated carbonyl group on the reducing agents was influenced by the ring size and steric factor. Cholesterol (cholest-5-en-3beta-ol) and dehydroepiandrosterone (DHEA) were oxidized with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone to form 1,4,6-cholestatrien-3-one and 1,4,6-androstatriene-3,17-dione. They were reduced with NaBH(4), lithium tri-sec-butylborohydride (l-Selectride), LiAlH(4), 9-borabicyclo[3.3.1]nonane (9-BBN), lithium triethylborohydride (Super-hydride), and BH(3) x (CH(3))(2)S in various conditions, respectively. Reduction of 1,4,6-cholestatrien-3-one and 1,4,6-androstatriene-3,17-dione by NaBH(4) (4 equiv.) produced 4,6-cholestadien-3beta-ol and 4,6-androstadiene-3beta,17beta-diol, respectively. Reduction by l-Selectride (12 equiv.) afforded 4,6-cholestadien-3alpha-ol and 4,6-androstadiene-3alpha,17beta-diol, chemoselectively. Reaction with Super-hydride (12 equiv.) produced 4,6-cholestadien-3-one and 3-oxo-4,6-androstadien-17beta-ol. Reduction of 1,4,6-cholestatrien-3-one by 9-BBN (14 equiv.) produced 1,4,6-cholestatrien-3alpha-ol, but 1,4,6-androstatriene-3,17-dione was not reacted with 9-BBN in the reaction conditions. Reaction of LiAlH(4) (6 equiv.) formed 4,6-cholestadien-3beta-ol and 3-oxo-1,4,6-androstatrien-17beta-ol. Reduction of 1,4,6-cholestatrien-3-one by BH(3) x (CH(3))(2)S (11 equiv.) gave cholestane as major compound and unlike reactivity of cholesterol, 1,4,6-androstatriene-3,17-dione by 8 equiv. of BH(3) x (CH(3))(2)S formed 3-oxo-1,4,6-androstatrien-17beta-ol. LiAlH(4) and BH(3) x (CH(3))(2)S showed relatively low chemoselectivity.     

Conversion of androstenedione to 3 beta-hydroxy-5-androsten-17-one and 3 beta-hydroxy-4-androsten-17-one by the testicular microsomal fraction of Sprague-Dawley rats

When androstenedione was incubated with testicular microsomes of Sprague-Dawley rats in the presence of reduced nicotinamide-adenine dinucleotide (NADH), unknown metabolites were produced, in addition to testosterone and 7 alpha-hydroxyandrostenedione. The metabolites were identified as 3 beta-hydroxy-4-androsten-17-one and 3 beta-hydroxy-5-androsten-17-one (3:1) by biochemical and radiochemical methods. These results confirmed the occurrence of the reverse reactions from androstenedione to 3 beta-hydroxy-4-androsten-17-one and 3 beta-hydroxy-5-androsten-17-one catalyzed by the 3 beta-hydroxysteroid dehydrogenase and 5-ene-4-ene isomerase in the microsomal fraction of Sprague-Dawley rat testes.     

Studies on Bacillus stearothermophilus. Part IV. Influence of enhancers on biotransformation of testosterone

The impact of chemical enhancers on the biotransformation of testosterone has been exploited. Application of crude cell concentrates to produce Bacillus stearothermophilus-mediated bioconversion of testosterone at 65 degrees C for 72 h has been examined. After incubation, the xenobiotic substrate was added to the concentrated whole cell suspensions. The enhancer molecules were included in the whole cell suspension. The resultant products, after extraction into an organic solvent, were purified by thin layer chromatography and identification was carried out through spectroscopic data. Five steroid metabolites 9,10-seco-4-androstene-3,9,17-trione, 5alpha-androstan-3,6,17-trione, 17beta-hydroxy-5alpha-androstan-3,6-dione, 3beta,17beta-dihydroxyandrost-4-ene-6-one and 17beta-hydroxyandrost-4,6-diene-3-one were identified as biotransformation products of testosterone. A possible biosynthetic route for these bioconversion products is postulated.