Kinetic competence of an externally generated dienol intermediate with steroid isomerase.

The putative intermediate dienol (2) in the steroid isomerase (KSI) catalyzed conversion of 5-androstene-3,17-dione (1) to 4-androstene-3,17-dione (3) has been independently generated and tested as a substrate for KSI. At pH 7, dienol 2 is converted by KSI to a mixture of 1 (46%) and 3 (54%). The apparent second-order rate constant for reaction of 2 with KSI to produce 3 (kappa cat/Km = 2.3 x 10(8) M-1 s-1) is similar to that for reaction of 1 with KSI (kappa cat/Km = 2.1 x 10(8) M-1 s-1), demonstrating that 2 is kinetically competent. Isomerization of 1 by KSI in D2O gives only 5% of solvent deuterium incorporated into the product 3. When 2 reacts with KSI in D2O, and the product 3 is isolated (from direct reaction of 2 and from subsequent conversion of the 1 initially formed), ca. 80 atom % deuterium is located at C-6 beta, confirming that protonation of the dienol by KSI occurs at the same face as the proton transfer in the KSI catalyzed reaction of 1 to 3.     

Human glutathione transferase A3-3, a highly efficient catalyst of double-bond isomerization in the biosynthetic pathway of steroid hormones.

The cDNA of a novel human glutathione transferase (GST) of the Alpha class was cloned, and the corresponding protein, denoted GST A3-3, was heterologously expressed and characterized. GST A3-3 was found to efficiently catalyze obligatory double-bond isomerizations of Delta(5)-androstene-3,17-dione and Delta(5)-pregnene-3,20-dione, precursors to testosterone and progesterone, respectively, in steroid hormone biosynthesis. The catalytic efficiency (k(cat)/K(m)) with Delta(5)-androstene-3,17-dione was determined as 5 x 10(6) m(-1) s(-1), which is considerably higher than with any other GST substrate tested. The rate of acceleration afforded by GST A3-3 is 6 x 10(8) based on the ratio between k(cat) and the rate constant for the nonenzymatic isomerization of Delta(5)-androstene-3,17-dione. Besides being high in absolute numbers, the k(cat)/K(m) value of GST A3-3 exceeds by a factor of approximately 230 that of 3beta-hydroxysteroid dehydrogenase/isomerase, the enzyme generally considered to catalyze the Delta(5)-Delta(4) double-bond isomerization. Furthermore, GSTA3-specific polymerase chain reaction analysis of cDNA libraries from various tissues showed a message only in those characterized by active steroid hormone biosynthesis, indicating a selective expression of GST A3-3 in these tissues. Based on this finding and the high activity with steroid substrates, we propose that GST A3-3 has evolved to catalyze isomerization reactions that contribute to the biosynthesis of steroid hormones.     

A theoretical model of the catalytic mechanism of the Delta5-3-ketosteroid isomerase reaction

The present paper describes a theoretical approach to the catalytic reaction mechanism involved in the conversion of 5-androstene-3,17-dione to 4-androstene-3,17-dione. The model incorporates the side chains of the residues tyrosine (Tyr(14)), aspartate (Asp(38)) and aspartic acid (Asp(99)) of the enzyme Delta(5)-3-ketosteroid isomerase (KSI; EC 5.3.3.1). The reaction involves two steps: first, Asp(38) acts as a base, abstracting the 4beta-H atom (proton) from C-4 of the steroid to form a dienolate as the intermediate; next, the intermediate is reketonized by proton transfer to the 6beta-position. Each step goes through its own transition state. Functional groups of the Tyr(14) and Asp(99) side chains act as hydrogen bond donors to the O1 atom of the steroid, providing stability along the reaction coordinate. Calculations were assessed at high level Hartree-Fock theory, using the 6-31G(*) basis set and the most important physicochemical properties involved in each step of the reaction, such as total energy, hardness, and dipole moment. Likewise, to explain the mechanism of reaction, highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), atomic orbital contributions to frontier orbitals formation, encoded electrostatic potentials, and atomic charges were used. Energy minima and transition state geometries were confirmed by vibrational frequency analysis. The mechanism described herein accounts for all of the properties, as well as the flow of atomic charges, explaining both catalytic mechanism and proficiency of KSI.     

The role of glutathione in the isomerization of delta 5-androstene-3,17-dione catalyzed by human glutathione transferase A1-1

Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Delta(5)-androstene-3,17-dione (AD) into Delta(4)-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited catalytic effect. S-Alkylglutathione derivatives do not promote the reaction, and the pH dependence of the isomerization indicates that the glutathione thiolate serves as a base in the catalytic mechanism. Mutation of the active-site Tyr(9) into Phe significantly decreases the steady-state kinetic parameters, alters their pH dependence, and increases the pK(a) value of the enzyme-bound glutathione thiol. Thus, Tyr(9) promotes the reaction via its phenolic hydroxyl group in protonated form. GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than GST A2-2 and GST A4-4, both having a glutathione cofactor and an active-site Tyr(9) residue. The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction. The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3beta-hydroxysteroid dehydrogenase.     

pH dependence of the kinetic parameters for 3-oxo-delta 5-steroid isomerase. Substrate catalysis and inhibition by (3S)-spiro[5 alpha-androstane-3,2'-oxiran]-17-one

The pH-rate profiles for kcatobsd and (kcat/KM)obsd at 25.0 degrees C have been measured for 3-oxo-delta 5-steroid isomerase by using 5-androstene-3,17-dione (2), 5-pregnene-3,20-dione (3), and 5(10)-estrene-3,17-dione (4) as substrates. Results from the nonsticky substrate 4 suggest values for the pK of a catalytically important group on the free enzyme (pKE) of 4.57 and the pK of the same group in the enzyme-substrate complex of 4.74. For the sticky substrates 2 and 3, pKES is ca. 4.75 and 5.5, respectively. Analysis of the (kcat/KM)obsd vs. pH profile for 2 reveals that the intermediate E X S complex decomposes to products at a rate similar to its reversion to E + S. The pH-rate profile for inhibition of the isomerase by (3S)-spiro-[5 alpha-androstane-3,2'-oxiran]-17-one (7 beta) shows values for pKE of 4.75 and pKEI of 4.90. The similarity of the pH-rate profiles for isomerization of 4 and inhibition by 7 beta suggests that both reactions may be governed by the ionization state of the same carboxyl group of the enzyme.     

Role of reduced glutathione in the delta(5)-3-kitosteroid isomerase reaction of liver.

Partially purified rat liver Δ⁵-3-ketosteroid isomerase (EC 5.3.3.1) is profoundly and specifically activated by reduced glutathione (GSH). This stimulating effect shows normal saturating kinetics, and both K_m and V_max are pH-dependent. The binding of GSH is independent of the concentration of Δ⁵-androstene-3,17-dione, whereas the K_m for Δ⁵-androstene-3,17-dione is markedly reduced by saturating levels of GSH. The same catalytic site appears to isomerize both Δ⁵-androstene-3,17-dione and Δ⁵-pregnene-3,20-dione. Several steroidal inhibitors compete with Δ⁵-androstene-3,17-dione, whereas $\text{S}$-methyl-glutathione competes with GSH. This activation of Δ⁵-3-ketosteroid isomerase is also observed in the livers of other species (calf, guinea pig, human), and represents a hitherto unrecognized function of reduced glutathione.     

Differences between bovine and human steroid double-bond isomerase activities of Alpha-class glutathione transferases selectively expressed in steroidogenic tissues

Bovine glutathione transferase A1-1 (bGST A1-1) and human GST A3-3 (hGST A3-3) share both high amino acid sequence similarity and selective expression in steroidogenic organs. hGST A3-3 is the most efficient steroid isomerase known in mammals, and is thought to catalyze isomerization reactions in the biosynthesis of steroid hormones. We observed that four out of five residues essential to the high steroid isomerase activity of hGST A3-3 are conserved in bGST A1-1. The bovine GST was cloned, heterologously expressed, and purified to homogeneity. Its specific activity towards classical GST substrates and two steroids, Delta(5)-androstene-3,17-dione and Delta(5)-pregnene-3,20-dione, was studied, and the steady-state kinetic parameters with the steroids were determined. We find that bGST A1-1 exhibits enzymatic activities comparable to those of hGST A3-3 towards non-steroid substrates. However, the bovine enzyme had 100 times lower catalytic efficiency in steroid isomerization reactions than the human GST. Nevertheless, bGST A1-1 was found as efficient as bovine 3beta-hydroxysteroid dehydrogenase as a steroid isomerase. We discuss likely reasons for the contrasting steroid isomerase activities of bGST A1-1 and hGST A3-3, and alternative roles of bGST A1-1.     

Catalysis of the isomerization of Δ5-3-ketosteroids to Δ4-3-ketosteroids by primary amines: Evidence for an imine intermediate

The isomerization of 5-androstene-3,17-dione and 17β-hydroxy-5-androstene-3-one to 4-androstene-3,17-dione and 17β-hydroxy-4-androstene-3-one, respectively, is catalyzed by primary amines. In the case of the isomerization catalyzed by glycylglycine the reaction proceeds through an intermediate which absorbs maximally at 275 nm. Based on spectral similarities to appropriate model compounds and structural analysis of the intermediate after its reduction by sodium borohydride, the intermediate has been tentatively identified as the Δ⁴-3-imine.     

Structure-based reconstruction of a Mycobacterium hypothetical protein into an active Δ5–3-ketosteroid isomerase

Protein engineering based on structure homology holds the potential to engineer steroid-transforming enzymes on demand. Based on the genome sequencing analysis of industrial Mycobacterium strain HGMS2 to produce 4-androstene-3,17-dione (4-AD), three hypothetical proteins were predicted as putative Δ⁵–3-ketosteroid isomerases (KSIs) to catalyze an intramolecular proton transfer involving the transformation of 5-androstene-3,17-dione (5-AD) into 4-AD, which were defined as mKSI228, mKSI291 and mKSI753. Activity assays indicated that mKSI228 and mKSI291 exhibited weak activity, as low as 0.7% and 1.5%, respectively, of a well-studied and highly active KSI from Pseudomonas putida KSI (pKSI), while mKSI753 had no activity similar to Mycobacterium tuberculosis KSI (mtKSI). Although the 3D structures of the putative mKSIs were homologous to pKSI, their amino acid sequences were significantly different from those of pKSI and tKSI. Thus, by use of these two KSIs as homology models, we were able to convert the low-active mKSI291 into a high-active active KSI by site-directed mutagenesis. On the other hand, an X-ray crystallographic structure of mKSI291 identified a water molecule in its active site. This unique water molecule might function as a bridge to connect Ser-OH, Tyr57-OH and C3O of the intermediate form a hydrogen-bonding network that was responsible for its weak activity, compared with that of mtKSI. Our results not only demonstrated the use of a protein engineering approach to understanding KSI catalytic mechanism, but also provided an example for engineering the catalytic active sites and gaining a functional enzyme based on homologous structures.     

Active-site residues governing high steroid isomerase activity in human glutathione transferase A3-3

Glutathione transferase (GST) A3-3 is the most efficient human steroid double-bond isomerase known. The activity with Delta(5)-androstene-3,17-dione is highly dependent on the phenolic hydroxyl group of Tyr-9 and the thiolate of glutathione. Removal of these groups caused an 1.1 x 10(5)-fold decrease in k(cat); the Y9F mutant displayed a 150-fold lower isomerase activity in the presence of glutathione and a further 740-fold lower activity in the absence of glutathione. The Y9F mutation in GST A3-3 did not markedly decrease the activity with the alternative substrate 1-chloro-2,4-dinitrobenzene. Residues Phe-10, Leu-111, and Ala-216 selectively govern the activity with the steroid substrate. Mutating residue 111 into phenylalanine caused a 25-fold decrease in k(cat)/K(m) for the steroid isomerization. The mutations A216S and F10S, separate or combined, affected the isomerase activity only marginally, but with the additional L111F mutation k(cat)/K(m) was reduced to 0.8% of that of the wild-type value. In contrast, the activities with 1-chloro-2,4-dinitrobenzene and phenethylisothiocyanate were not largely affected by the combined mutations F10S/L111F/A216S. K(i) values for Delta(5)-androstene-3,17-dione and Delta(4)-androstene-3,17-dione were increased by the triple mutation F10S/L111F/A216S. The pK(a) of the thiol group of active-site-bound glutathione, 6.1, increased to 6.5 in GST A3-3/Y9F. The pK(a) of the active-site Tyr-9 was 7.9 for the wild-type enzyme. The pH dependence of k(cat)/K(m) of wild-type GST A3-3 for the isomerase reaction displays two kinetic pK(a) values, 6.2 and 8.1. The basic limb of the pH dependence of k(cat) and k(cat)/K(m) disappears in the Y9F mutant. Therefore, the higher kinetic pK(a) reflects ionization of Tyr-9, and the lower one reflects ionization of glutathione. We propose a reaction mechanism for the double-bond isomerization involving abstraction of a proton from C4 in the steroid accompanied by protonation of C6, the thiolate of glutathione serving as a base and Tyr-9 assisting by polarizing the 3-oxo group of the substrate.     

Targeting human glutathione transferase A3-3 attenuates progesterone production in human steroidogenic cells

hGSTA3-3 (human Alpha-class glutathione transferase 3-3) efficiently catalyses steroid Delta(5)-Delta(4) double-bond isomerization in vitro, using glutathione as a cofactor. This chemical transformation is an obligatory reaction in the biosynthesis of steroid hormones and follows the oxidation of 3beta-hydroxysteroids catalysed by 3beta-HSD (3beta-hydroxysteroid dehydrogenase). The isomerization has commonly been ascribed to a supplementary function of 3beta-HSD. The present study is the first to provide evidence that hGSTA3-3 contributes to this step in steroid hormone biosynthesis in complex cellular systems. First, we find glutathione-dependent Delta(5)-Delta(4) isomerase activity in whole-cell extracts prepared from human steroidogenic cells. Secondly, effective inhibitors of hGSTA3-3 dramatically decrease the conversion of Delta(5)-androstene-3,17-dione into Delta(4)-androstene-3,17-dione in cell lysates. Thirdly, we show that RNAi (RNA interference) targeting hGSTA3-3 expression decreases by 30% the forskolin-stimulated production of the steroid hormone progesterone in a human placental cell line. This effect is achieved at low concentrations of two small interfering RNAs directed against distinct regions of hGSTA3-3 mRNA, and is weaker in unstimulated cells, in which hGSTA3-3 expression is low. The results concordantly show that hGSTA3-3 makes a significant contribution to the double-bond isomerization necessary for steroid hormone biosynthesis and thereby complements the indispensable 3beta-hydroxysteroid oxidoreductase activity of 3beta-HSD. The results indicate that the lower isomerase activity of 3beta-HSD is insufficient for maximal rate of cellular sex hormone production and identify hGSTA3-3 as a possible target for pharmaceutical intervention in steroid hormone-dependent diseases.     

Affinity labeling of rat glutathione S-transferase isozyme 1-1 by 17beta -iodoacetoxy-estradiol-3-sulfate

Rat liver glutathione S-transferase, isozyme 1-1, catalyzes the glutathione-dependent isomerization of Delta(5)-androstene-3,17-dione and also binds steroid sulfates at a nonsubstrate inhibitory steroid site. 17beta-Iodoacetoxy-estradiol-3-sulfate, a reactive steroid analogue, produces a time-dependent inactivation of this glutathione S-transferase to a limit of 60% residual activity. The rate constant for inactivation (k(obs)) exhibits a nonlinear dependence on reagent concentration with K(I) = 71 microm and k(max) = 0.0133 min(-1). Complete protection against inactivation is provided by 17beta-estradiol-3,17-disulfate, whereas Delta5-androstene-3,17-dione and S-methylglutathione have little effect on k(obs). These results indicate that 17beta-iodoacetoxy-estradiol-3-sulfate reacts as an affinity label of the nonsubstrate steroid site rather than of the substrate sites occupied by Delta5-androstene-3,17-dione or glutathione. Loss of activity occurs concomitant with incorporation of about 1 mol 14C-labeled reagent/mol enzyme dimer when the enzyme is maximally inactivated. Isolation of the labeled peptide from the chymotryptic digest shows that Cys(17) is the only enzymic amino acid modified. Covalent modification of Cys(17) by 17beta-iodoacetoxy-estradiol-3-sulfate on subunit A prevents reaction of the steroid analogue with subunit B. These results and examination of the crystal structure of the enzyme suggest that the interaction between the two subunits of glutathione S-transferase 1-1, and the electrostatic attraction between the 3-sulfate of the reagent and Arg(14) of subunit B, are important in binding steroid sulfates at the nonsubstrate steroid binding site and in determining the specificity of this affinity label.     

Steroid metabolism in gonads of turtle embryos as a function of the incubation temperature of eggs

In embryos of many reptiles, the sexual differentiation of gonads is temperature-dependent. In the turtle Emys orbicularis, all individuals become phenotypic males at 25 degrees C, whereas 100% phenotypic females are obtained at 30 degrees C. Steroid metabolism in embryonic gonads was studied at both temperatures, during and after the thermosensitive period for sexual differentiation. Pools of gonads were incubated for various times, with 3 beta-hydroxy-5-pregnen-20-one (pregnenolone), progesterone, dehydroepiandrosterone or 4-androstene-3,17- dione as substrates. The analysis of metabolites combined two successive chromatographies (HPLC and TLC) and autoradiography. Conversion of pregnenolone to progesterone and of dehydroepiandrosterone to 4-androstene-3,17-dione was more important in testes at 25 degrees C than in ovaries at 30 degrees C. In ovaries, a large amount of 5-pregnene- 3 beta,20 beta-diol was formed from pregnenolone, and 5-androstene-3 beta,17 beta-diol was produced from dehydroepiandrosterone. In both testes and ovaries, 5 alpha-pregnane and 5 alpha-androstane derivatives were the main metabolites obtained from progesterone and 4-androstene-3,17-dione, respectively. Progesterone was also converted to 20 beta-hydroxy-4-pregnen-3-one. Dehydroepiandrosterone and 4-androstene-3,17-dione were also metabolized into 11 beta-hydroxy-4-androstene-3,17-dione (only in testes), testosterone, 11 beta,17 beta-dihydroxy-4-androstene-3-one, 17 beta-hydroxy-4-androstene-3,11-dione (low amounts in testes, traces in ovaries), 17 alpha-hydroxy-4-androstene-3-one, estrone and estradiol-17 beta (traces).     

Crystal structure of human glutathione S-transferase A3-3 and mechanistic implications for its high steroid isomerase activity

The crystal structure of human class alpha glutathione (GSH) S-transferase A3-3 (hGSTA3-3) in complex with GSH was determined at 2.4 A. Despite considerable amino acid sequence identity with other human class alpha GSTs (e.g., hGSTA1-1), hGSTA3-3 is unique due to its exceptionally high steroid double bond isomerase activity for the transformation of Delta(5)-androstene-3,17-dione (Delta(5)-AD) to Delta(4)-androstene-3,17-dione. A comparative analysis of the active centers of hGSTA1-1 and hGSTA3-3 reveals that residues in positions 12 and 208 may contribute to their disparate isomerase activity toward Delta(5)-AD. Substitution of these two residues of hGSTA3-3 with the corresponding residues in hGSTA1-1 followed by kinetic characterization of the wild-type and the mutant enzymes supported this prediction. On the basis of our model of the hGSTA3-3.GSH.Delta(5)-AD ternary complex and available biochemical data, we propose that the thiolate group of deprotonated GSH (GS(-)) serves as a base to initiate the reaction by accepting a proton from the steroid and the nonionized hydroxyl group of catalytic residue Y9 (HO-Y9) functions as part of a proton-conducting wire to transfer a proton back to the steroid. Residue R15 may function to stabilize the deprotonated thiolate group of GSH (GS(-)), and a GSH-bound water molecule may donate a hydrogen bond to the 3-keto group of Delta(5)-AD and thus help the thiolate of GS(-) to initiate the proton transfer and the subsequent stabilization of the reaction intermediate.     

Development of a sensitive and specific new plasma 4-androstene-3,17- dione time-resolved fluoroimmunoassay (TR-FIA)

We describe, for the first time to our knowledge, the development of a new, non-isotopic time resolved-fluoroimmunoassay of 4-androstene-3,17-dione in plasma or serum. This steroid exhibits a key role in steroid metabolism and is often assayed in the investigation of various pathologic endocrine states. Most of the 4-androstene-3,17-dione immunoassays are performed using a radioactive tracer. We synthesized a biotinylated 4-androstene-3,17-dione tracer from 4-androstene-3,17-dione-3-carboxymethyloxime by acylation of biotinylaminopropylammonium trifluoroacetate. A specific rabbit anti 6-hemisuccinate-4-androstene-3,17-dione/BSA was indirectly bound via an anti-rabbit sheep antibody immobilized on microtiter plate wells. The amount of biotinylated-4-androstene-3,17-dione tracer was then measured by adding streptavidin-europium, and the europium fluorescence was quantified by time resolved-fluorescence (TR-FIA, Delfia System). The plasma 4-androstene-3,17-dione-levels measured with this non-isotopic assay were compared to those measured with a radioimmunoassay previously published. In both cases, the same anti-4-androstene-3,17-dione antibody was used, and the assays were performed after an extraction step and a chromatographic step. The results obtained by the two methods were virtually the same. However, the main advantages of the new plasma 4-androstenedione-3,17-dione time-resolved-fluorescence immunoassay were its greater sensitivity than radioimmunoassay and its higher precision.     

The catalytic power of ketosteroid isomerase investigated by computer simulation

Ketosteroid isomerase (KSI) catalyzes the isomerization of Delta(5)-3-ketosteroids and Delta(4)-3-ketosteroids at very high rates. Here we examine the principles underlying the catalytic efficiency of KSI by computer simulations using the empirical valence bond method in combination with molecular dynamics free energy perturbation simulations. The simulations reproduce available kinetic and structural data very well and allow us to examine several features of the catalytic mechanism in detail. It is found that about 60% of the rate enhancement is due to stabilization of the negatively charged dienolate intermediate by hydrogen bonding. The critical H-bond between Tyr16 and the intermediate is found to be a normal ionic H-bond with the preferred proton location on the tyrosine residue. The remaining 40% of the catalytic effect originates from a reduction of the reorganization energy of the reaction. The possibility of an active site water molecule occupying the empty cavity adjacent to the catalytic base (Asp40) is also addressed. The existence of such a water molecule could explain how the enzyme manages to maintain a low pK(a) for the general base residue.     

Immobilization of Rhodococcus equi DSM 89-133 onto porous celite beads for cholesterol side-chain cleavage

Summary Rhodococcus equi cells immobilized onto porous celite beads were active in side-chain cleavage of cholesterol. The effect of bead size, adsorption time and washing cycles were examined. The degradation of cholesterol side-chain using adsorbed cells was studied in batch and semi-continuous systems. Compared to free cells (80 mol%), the end-product (1,4-androstadiene-3,17-dione and 4-androstene-3,17-dione) profile was lower (70 mol%) with the celite-adsorbed system. Correspondence to: B. N. Johri     

Synthesis and evaluation of analogues of 4-androstene-3,17-dione as aromatase inhibitors

Twenty-three synthetic analogues of 4-androstene-3,17-dione (androstenedione) have been evaluated as inhibitors of human placental microsomal aromatase enzyme. Among the most potent of these compounds were the 4-hydroxy, 6 alpha-fluoro, 6 beta-fluoro, and 4-fluoroandrostenediones and 4-fluoro-19-nor-4-androstene-3,17-dione. 4-Hydroxy-4-androstene-3,17-dione (4HAD) is an irreversible inhibitor of aromatase in vitro, whereas the four fluoro analogues are reversible inhibitors. 4HAD and 4-fluoro-4-androstene-3,17-dione caused significant regression of the nitrosomethylurea-induced mammary tumor in rats, but the other fluoro derivatives were inactive.     

In vitro metabolism of 4-androstene-3,17-dione and testosterone by rat submaxillary gland.

Tritiated 4-androstene-3,17-dione and testosterone were incubated with submaxillary gland homogenates of male and female rats. The metabolism was predominately reductive. In 15 and 180 min incubations submaxillary tissue converted 4-androstene-3,17-dione chiefly to androsterone. Less testosterone, 17 beta-hydroxy-5 alpha-androstan-3-one, 5 alpha-androstane-3,17-dione, 5 alpha-androstane-3 alpha, 17 beta-diol, and 4-androstene-3 alpha, 17 beta-diol were also identified. Testosterone was converted to the same products plus 4-androstene-3,17-dione. 5 alpha-Androstane-3 alpha, 17 beta-diol was the major testosterone metabolite. Qualitatively the metabolism by male and female submaxillary gland was similar.     

An improved radioimmunoassay for 4-androstene-3,17-dione

A radioimmunoassay using an antiserum produced against 6β-hydroxy-4-androstene-3,17-dione-6-succinyl-BSA conjugate is described which permits the rapid determination of 4-androstene-3,17-dione in multiple serum samples that are purified by column chromatography on neutral alumina. Steroids which reacted significantly with the antiserum were found to be 5α-androstane-3,17-dione, 5β-androstane-3,17-dione and 6β-hydroxy-4-androstene-3,17-dione. After column chromatography on alumina, however, the only significantly cross-reacting steroids were the 5α and 5β-androstane-3,17-diones, while cross-reactivity from other steroids was reduced to less than 1%.