Km For Testosterone Research Articles

Expression in Escherichia Coli, Purification, and Functional Reconstitution of Human Steroid 5α-Reductases.

The potent androgen 5α-dihydrotestosterone irreversibly derives from testosterone via the activity of steroid 5α-reductases (5αRs). The major 5αR isoforms in most species, 5αR1 and 5αR2, have not been purified to homogeneity. We report here the heterologous expression of polyhistidine-tagged, codon-optimized human 5αR1 and 5αR2 cDNAs in Escherichia coli. A combination of the nonionic detergents Triton X-100 and Nonidet P-40 enabled solubilization of these extremely hydrophobic integral membrane proteins and facilitated purification with affinity and cation-exchange chromatography methods. For functional reconstitution, we incorporated the purified isoenzymes into Triton X-100-saturated dioleoylphosphatidylcholine liposomes and removed excess detergent with polystyrene beads. Kinetic studies indicated that the 2 isozymes differ in biochemical properties, with 5αR2 having a lower apparent Km for testosterone, androstenedione, progesterone, and 17-hydroxyprogesterone than 5αR1; however, 5αR1 had a greater capacity for steroid conversion, as reflected by a higher Vmax than 5αR2. Both enzymes preferred progesterone as substrate over other steroids, and the catalytic efficiency of purified reconstituted 5αR2 exhibited a sharp pH optimum at pH 5. Intriguingly, we found that the prostate-cancer drug-metabolite 3-keto-∆ 4-abiraterone is metabolized by 5αR1 but not 5αR2, which may serve as a structural basis for isoform selectivity and inhibitor design. The functional characterization results with the purified reconstituted isoenzymes paralleled trends obtained with HEK-293 cell lines stably expressing native 5αR1 and 5αR2. Access to purified human 5αR1 and 5αR2 will advance studies of these important enzymes and might help to clarify their contributions to steroid anabolism and catabolism.

Structure-function studies of human 5-alpha reductase type 2 using site directed mutagenesis

Site directed mutagenesis of human steroid 5α-reductase types 1 (5AR1) and 2 (5AR2) has been used to identify residues involved in inhibitor/substrate binding by 5AR2. Replacing residues 21–24 (GALA) in 5AR2 with the analogous residues 26–29 (AVFA) from 5AR1 did not significantly alter either the Km for testosterone or the Ki for the competitive inhibitor Finasteride. Replacement of AVFA in 5AR1 with GALA from 5AR2 however, significantly decreased the Km and increased the resistance to Finasteride. These findings confirm that 5AR1 residues 26–29 are involved in inhibitor/substrate binding but suggest residues 21–24 of 5AR2 are not. Replacing residues 20–29 (QCAVGCAVFA) of 5AR1 with the analogous residues 15–24 (ATLVALGALA) from 5AR2, changed the Km and Ki to values approaching those for wild type 5AR2. Replacing residues VAL in wild type 5AR2 with VGC from 5AR1 did not change Km or Ki but replacing ATL in 5AR2 with QCA from 5AR1 significantly decreased the Km and increased the resistance to Finasteride. Conversely, replacing QCA with ATL in 5AR1 containing GALA in place of AVFA, increased the Km and decreased resistance to Finasteride. These findings indicate residues 15–17 of human 5AR2 participate in inhibitor/substrate binding whereas residues 18–20 do not.

Testosterone dehydrogenase activity in koala liver: characterisation of cofactor and steroid substrate differences

We have studied the hepatic microsomal 17β-hydroxysteroid dehydrogenase (17β-HSD) capacity of koala ( Phascolarctos cinereus) and tammar wallaby ( Macropus eugenii). A detailed comparison of the activity in hepatic fractions from koala and rat was made. Hepatic microsomal NADP-supported 17β-HSD activity was significantly higher in koala (11.64±3.35 nmoles/mg protein/min), (mean±S.D.) than in tammar wallaby liver (1.52±0.79 nmoles/mg protein/min). However, when NAD was utilised as cofactor the activity was similar in both marsupial species (2.83±2.03 nmoles/mg protein/min, koala; 0.70±0.71 nmoles/mg protein/min, tammar wallaby). Data for rat indicated a cofactor preference for NAD rather than NADP (17.94±6.40 nmoles/mg protein/min, NAD; 2.18±1.04 nmoles/mg protein/min, NADP). Michaelis–Menten parameters for the kinetics of 17β-HSD testosterone oxidation by NADP and NAD were determined in the koala. The Km for testosterone was of the order of 10.0–24.0 μM ( n=6) irrespective of the cofactor used, whilst the Km for NADP was 0.28–0.43 μM ( n=2) and for NAD was 13.9–18.5 μM ( n=2). 17β-estradiol was found to be an inhibitor of both NAD- and NADP- supported 17β-HSD activity. These findings indicate that NADP-mediated, but not NAD-mediated testosterone dehydrogenation is a major pathway of steroid biotransformation in koala liver; the reaction is less extensive in fractions from wallaby, human and rat. Such species-related differences in cofactor preference may contribute along with species differences in gene expression to observed rates of 17β-HSD activity in mammals.

Site-directed mutagenesis studies of the NADPH-binding domain of rat steroid 5α-reductase (isozyme-1) I: Analysis of aromatic and hydroxylated amino acid residues 1

Previous studies have shown that the reduced nicotinamide adenine dinucleotide phosphate (NADPH)- binding domain of rat liver microsomal steroid 5α-reductase isozyme-1 (r5αR-1) is in a highly conserved region of the polypeptide sequence (residues 160–190). In this study, we investigated, by site-directed mutagenesis, the role of hydroxylated and aromatic amino acids within the NADPH-binding domain. The r5αR-1 cDNA was cloned into a pCMV vector, and the double strand site-directed mutagenesis method was used to create mutants Y179F, Y179S, Y189F, Y189S, S164A, S164T, and Y187F, which were subsequently expressed in COS-1 cells. Kinetic studies of the expressed enzymes showed that the mutation Y179F resulted in an ∼40-fold increase in the Km for NADPH versus wild-type, with only a 2-fold increase in the Km for testosterone. The mutants Y189F and S164A showed smaller increases (4 and 6-fold) in Kms for NADPH and no significant change in the Km for testosterone, whereas Y189S had kinetic properties similar to the wild-type r5αR-1. Mutants Y179S and S164T both resulted in inactive enzymes, whereas F187Y showed an ∼5-fold decrease in Km for NADPH and a significant increase (∼18-fold) in the Km for testosterone. The results suggest that the -OH functionality of Y179 is involved in cofactor binding, but is not essential for the activity of the enzyme, whereas the -OH functionalities of Y189 and S164 play lesser roles in cofactor binding to r5αR-1 and may not be required for enzyme activity. On the other hand, the residue F187 may be important for the binding of both NADPH and testosterone.

Influence of Glutathione on the Catalytic Activity of Reconstituted Cytochrome P450 3A4

The role of NADPH reductase and cytochrome b5on glutathione (GSH)-induced stimulation of P450 3A4 activity was investigated. GSH increased the Vmaxof testosterone 6β-hydroxylation without changing the Kmfor testosterone whereas it decreased the Kmfor NADPH-P450 reductase. Addition of cytochrome b5inhibited testosterone 6β-hydroxylation in the reconstituted system, depleting GSH, while it dramatically enhanced the rate of testosterone 6β-hydroxylation in the presence of GSH. Cumene hydroperoxide-mediated P450 3A4 activity, which is independent of NADPH-P450 reductase and cytochrome b5, was not affected by GSH. High concentration of GSH above 4 mM was inhibitory in the reconstituted systems. These results suggest that GSH increases the apparent affinity between P450 3A4 and NADPH-P450 reductase, and between P450 3A4 and cytochrome b5, but has no effect on the affinity between P450 3A4 and testosterone.

Androgen UDP-glucuronyl transferase activity is found primarily in liver in the rat.

UDP-glucuronyl transferase (UDPGT) activity was determined for androgens in tissue minces and microsomal fractions from the liver and extrahepatic tissues (kidney, skin, prostate, and preputial glands) of the male rat. Liver microsomes showed the highest UDPGT activity with each of the androgens tested (Vmax = 7, 3, and 10 nmol/minute/mg protein for testosterone, androsterone, and androstanediol, respectively). UDPGT activity (Vmax) for androstanediol in the liver was 10(2)-fold higher than in the kidney and 10(3)-fold higher than in the skin and prostate. UDPGT activity for androgens was not detected in microsomes from preputial glands. Furthermore, no body site distribution was found for androgen UDPGT activity in skin microsomes. The Michaelis-Menten constant (Km) for UDPGT in liver microsomes was 20.4, 12.2, and 2.2 microM, respectively, for testosterone, androstanediol, and androsterone. Kidney microsomes showed a Km of 19.4 and 26.9 microM, respectively, for androstanediol and androsterone. The Km for testosterone was very high in the kidney (138 microM), suggesting that it was a poor substrate. In microsomes from the skin and prostate, the Km was very high (range 43-162 microM) for all three androgen substrates, suggesting that these androgens were not the preferred substrates for UDPGT in these tissues. These results indicate that the liver was the main site of androgen UDPGT activity and the skin and prostate formed little, if any, androgen glucuronides. These results suggest that androstanediol glucuronide was formed primarily in the liver and may not be a reliable marker of peripheral androgen metabolism.

Baculovirus-directed expression of human prostatic steroid 5α-reductase 1 in an active form

In the human prostate, the enzyme steroid 5α-reductase (h5αR) catalyses the conversion of testosterone into the more potent androgen, dihydrotestosterone. Two distinct cDNAs coding for h5αR in the human prostate have been previously characterized. Enzyme h5αR1 shows a maximum activity at basic pH whereas h5αR2 has an acidic pH optimum activity. We report here the expression of the human steroid h5αR1 in a eukaryotic expression system : the baculovirus-directed-insect cell expression system. The full length cDNA was inserted into the Autographa californica nuclear polyhedrosis virus genome and expressed in Spodoptera frugiperda, Sf9, insect cells. Sf9 cells were infected with the recombinant baculovirus and homogenates used in h5αR activity assays by high pressure liquid chromatography showed that a catalytically active enzyme was produced. The recombinant enzyme showed an apparent Km for testosterone of 2.07 μM and a Vmax of 10.1 nmol of dihydrotestosterone/min/mg of protein. Recombinant h5αR1 activity was inhibited by specific h5αR inhibitors such as 4-MA (Ki = 2.6 nM). Subcellular distribution in Sf9 cells demonstrated that the enzyme was associated with the nuclear membrane.

Androgen UDP‐Glucuronyl Transferase Activity Is Found Primarily in Liver in the Rat

ABSTRACT: UDP‐glucuronyl transferase (UDPGT) activity was determined for androgens in tissue minces and microsomal fractions from the liver and extrahepatic tissues (kidney, skin, prostate, and preputial glands) of the male rat. Liver microsomes showed the highest UDPGT activity with each of the androgens tested (Vmax = 7, 3, and 10 nmol/minute/mg protein for testosterone, androsterone, and androstanediol, respectively). UDPGT activity (Vmax) for androstanediol in the liver was 102‐fold higher than in the kidney and 103‐fold higher than in the skin and prostate. UDPGT activity for androgens was not detected in microsomes from preputial glands. Furthermore, no body site distribution was found for androgen UDPGT activity in skin microsomes. The Michaelis‐Menten constant (Km) for UDPGT in liver microsomes was 20.4, 12.2, and 2.2 μ, respectively, for testosterone, androstanediol, and androsterone. Kidney microsomes showed a Km of 19.4 and 26.9 μ, respectively, for androstanediol and androsterone. The Km for testosterone was very high in the kidney (138 μ), suggesting that it was a poor substrate. In microsomes from the skin and prostate, the Km was very high (range 43–162 μ) for all three androgen substrates, suggesting that these androgens were not the preferred substrates for UDPGT in these tissues.These results indicate that the liver was the main site of androgen UDPGT activity and the skin and prostate formed little, if any, androgen glucuronides. These results suggest that androstanediol glucuronide was formed primarily in the liver and may not be a reliable marker of peripheral androgen metabolism.

Gonadotropic regulation of aromatase activity in the adult rat testis.

Aromatase activity during gonadotropin action in vivo and in vitro was examined in purified Leydig cells to further define the early effects of LH and for elucidation of the enzymatic processes involved in the development of late lesions of androgen biosynthetic pathway. Aromatase was measured by the tritiated water release method using [1 beta-3H]testosterone as substrate. The enzyme activity was proportional to the amount of cells (0.1-1.0 X 10(6] incubated, increased with the incubation time (2-60 min), was inhibited by androstatriendione (ED50, 5.0 microM), and showed a Km for testosterone of 1.69 microM. Aromatase activity was stimulated (10-20%; P less than 0.05) 1 h after treatment of rats with a single sc dose of 5 micrograms hCG. This activation preceded the late steroidogenic lesion at the site of 17 alpha-hydroxylase and 17,20-desmolase activity by 2-5 h. A RIA of improved sensitivity (0.5 pg) was developed to detect the very low cellular and secretory levels of estradiol. The testicular contents of testosterone and estradiol showed small increases (P less than 0.05) within 40 and 60 min after hCG treatment, respectively. Testicular testosterone levels reached a peak by 1 h after injection and preceded the peak level of estradiol formation by 2 h. After in vitro treatment of cultured Leydig cells with 100 ng hCG, the aromatase activity was significantly increased within 30 min (P less than 0.05) and then returned to control levels for up to 16 h of culture. A similar temporal pattern for enzyme activation was observed after treatment of cultures with 8-bromo-cAMP or forskolin (23-27% above control; P less than 0.05), while cholera toxin stimulated aromatase activity at 2 h. Net testosterone accumulation in the incubation medium increased 30 min after the hCG treatment and reached a plateau by 4 h. A small but significant increase in estradiol levels (P less than 0.05) was also observed at 30 min, remaining constant until 120 min, which was followed by a sharp rise parallel to that of testosterone. These results suggest that the estradiol-mediated desensitization of the Leydig cell observed after hCG administration is consistent with an early cAMP-dependent activation of aromatase and a further rise in estradiol formation due to increased substrate availability.

Microsomal 17beta-hydroxysteroid dehydrogenase of guinea pig liver: detergent solubilization and a comparison of kinetic and stability properties of bound and solubilized forms.

Abstract Microsomal 17β-hydroxy steroid dehydrogenase of guinea pig liver, solubilized in Triton X-100, was obtained in phospholipid-free form and physical parameters were estimated by agarose gel chromtography and density gradient centrifugation. The values for the sedimentation constant, s20,w = 5.2 s, partial specific volume, v = 0.78 ml/g , and Stokes radius, a = 66 A, indicated a molecular weight of 176,000 for the solubilized enzyme and were consistent with the presence of a significant amount of bound detergent. Triton X-100 was an inhibitor competitive with testosterone with a K1 of 0.11%. With solubilization the apparent km for testosterone was increased from 2.2 μM to 7.3 μM and the apparent KM for NAD+ reduced from 164 to 100 μM. The susceptibility to urea or trypsin inactivation was increased after solubilization. Enzymatic activity was stable for at least 48 h in 1% (v/v) Triton X-100 but was lost within 24 h in deoxycholate. The results show that phospholipids are not absolutely required for activity but the changes in kinetic and stability properties with solubilization are consistent with a role for membrane components or bound detergent in affecting the structure and catalytic properties of the enzyme.

The 17 β hydroxysteroïd NAD (P) oxidoreductases from female rabbit liver cytosol: separation and characterization

Summary DEAE cellulose chromatography achieved the separation of several 17 β hydroxysteroid NAD (P) oxidoreductase activities from rabbit liver cytosol; two of these activities differ strongly by their anionic character and electrophoretic mobility, as well as by their substrate or coenzyme specificity; the purification of these two fractions (A and C) was performed further. The fraction A contains a weakly anionic 17 β hydroxysteroid oxidoreductase. Its molecular weight is about 25 – 30 000 (sephadex G 100 filtration), both testosterone and estradiol are substrates; NAD or NADP are coenzymes, and the Km for testosterone is 5.10−4 M. From the second fraction (C) was separated a very anionic testosterone NADP oxidoreductase with high affinity for testosterone (Km 3 ± 0,5 10−6 M). The four isozymes shown by polyacrylamide gel electrophoresis are resolved into a single peptid chain of M.W. 36 000 by SDS polyacrylamide gel electrophoresis. The purified enzyme of fraction (C) exhibits a high 3α hydroxysteroid NADP oxidoreductase activity: experimental results are given showing electrophoretic and kinetic evidences for the identity of these two enzymes in fraction C of the adult female rabbit.