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PubMed1985

The in vitro metabolism of radioactive progesterone and testosterone by the gonads of the protandrous Rhabdosargus sarba at various sexual phases.

The in vitro steroidogenic capacity of the gonadal tissue in the protandrous Rhabdosargus sarba was studied. Testicular and ovarian tissues from various sexual phases were used either separately or combined. With progesterone as precursor, high yield of 5 beta-reduced metabolites, and no 11-ketotestosterone or 11 beta-hydroxytestosterone were found. The production of 5 beta-pregnan-3 alpha-ol-20-one and 5 beta-pregnane-3,20-dione was very high in incubations with testicular tissue from intersexes or males and was low in those with ovarian tissue only. The production of 17 alpha-hydroxyprogesterone was high in the female but was low in other sexual phases. With testosterone as precursor, 11-ketotestoterone and 11 beta-hydroxytestosterone were identified. The latter was the most abundant and prominent polar steroid in all incubations. 5 beta-Reductase activity was high in the male and relatively low in the female. 5 alpha-Reduced products, however, were absent. There was an increase in the production of androstenedione as the animal underwent sex reversal. The significance of this change in steroidogenesis in this protandrous fish is at present under investigation. Experimental results also indicated that in the intersexual gonad there may be interaction between the testicular component and the ovarian component in steroidgenesis.

Animals↗
PubMed1986

Steroid metabolism in the testes of the African catfish, Clarias gariepinus (Burchell), during spawning season, under natural conditions and kept in ponds.

Steroid metabolism in the testes of Clarias gariepinus, collected in the Hula nature reserve and a fish pond in North Israel during the spawning season, was studied in vitro by homogenate and tissue incubations with [3H]pregnenolone or [3H]androstenedione as precursors. It could be demonstrated that [3H]pregnenolone was mainly converted via progesterone and 17 alpha-hydroxyprogesterone into androstenedione, 11 beta-hydroxyandrostenedione, and 11 beta-hydroxytestosterone. The final products from [3H]androstenedione incubations, 11 beta-hydroxyandrostenedione, and 11 beta-hydroxytestosterone, confirm these findings. The major difference in steroid metabolism between wild and pond animals was a reduction in androgen synthesis in the first group, resulting in higher amounts of C21-steroids. Moreover, the synthesis of two steroid esters, including pregnenolone ester, could be established with tissue incubations of wild animals only. Steroid conjugates were hardly synthesized at all in the testes of C. gariepinus.

Androstenedione↗
PubMed1987

The plasma sex steroid profiles in the freshwater, sex-reversing teleost fish, Monopterus albus (Zuiew).

The plasma levels of both the free and conjugated forms of six sex steroids (androstenedione, testosterone, 11-oxotestosterone, 11 beta-hydroxytestosterone, estrone, and 17 beta-estradiol) were determined by radioimmunoassay combined with Celite chromatography in different sexual phases of the protogynous Monopterus albus throughout the reproductive cycle. The amounts of 11-oxotestosterone and 11 beta-hydroxytestosterone were found to be very low and variable in all the specimens investigated. No conjugated 17 beta-estradiol or free estrone was detected. Female individuals showed a prespawning rise of androstenedione which subsequently dropped to a low level in the spawning period. The estrogen levels in the female phase were found to be higher than those in all other sexual phases during the spawning period, but the testosterone level remained constant throughout the reproductive cycle in the female phase. The level of androstenedione was highest in the early intersexual and mid-intersexual phases during the postspawning/inactive period. Compared with the female specimens in the same reproductive period, the early intersexual individuals showed a higher level of 17 beta-estradiol, while the mid-intersexual animals showed a higher level of testosterone in the postspawned/inactive period. In the mid-intersexual phase, the levels of androstenedione, testosterone, 17 beta-estradiol, and estrone dropped progressively in relation to the seasonal reproductive cycle. The male fish had a constant level of androstenedione, estrone, and 17 beta-estradiol. However, the level of testosterone increased as the spawning period approached. The hormonal profile in the late intersexual phase was essentially similar to that in the male phase. The changes in the plasma levels of sex steroids in M. albus apparently were related to the maturation of the female and male sex tissues and to their seasonal reproductive cycle.

Androstenedione↗
PubMed1987

A radioimmunoassay study of the plasma levels of sex steroids in the protandrous, sex-reversing fish Rhabdosargus sarba (Sparidae).

By Celite chromatography and radioimmunoassay, the levels of free and conjugated forms of androstenedione, testosterone, 11-oxotestosterone, 11 beta-hydroxytestosterone, 17 beta-estradiol, and estrone have been determined in the plasma of the protandrous sex-reversing Rhabdosargus sarba at different sexual phases throughout the seasonal reproductive cycle. Free androstenedione showed a significant increase during the spawning period in the functional male phase, while a prespawning increase in both the free and the conjugated androstenedione was found in the female phase. The level of testosterone remained fairly constant throughout the reproductive cycle in all sexual phases except the intersexual phase, which was found to have a slight increase in the free testosterone level during the spawning period. Females showed a prespawning increase in free 17 beta-estradiol and conjugated estrone; the former dropped while the latter remained unchanged in the succeeding spawning period. A prespawning rise of conjugated 17 beta-estradiol was also found in the male specimens. The level of free estrone was undetectable in the plasma of Rhabdosargus. No variation in the levels of 11-oxotestosterone and 11 beta-hydroxytestosterone was found in the different sexual phases throughout the reproductive cycle. The present study showed that in general the steroid patterns in the male and intersexual phases were basically similar, except perhaps that the levels of conjugated 17 beta-estradiol, conjugated androstenedione, and free testosterone were slightly higher in the intersex than in the male during the spawning period. The changes in the plasma levels of sex steroids were correlated with the structural transformation in the gonad and the seasonal cycle of the fish. The results were analyzed with special references to the phenomenon of natural sex reversal and to the endocrine patterns known in other teleost fishes.

Androstenedione↗
PubMed1983

The unusual estrogen-binding protein (UEBP) of male rat liver: structural determinants of ligands.

The unusual estrogen-binding protein (UEBP) found in a male rat liver is a sex dependent protein which differs from other known receptor and transport proteins by the high lability of its complexes with estradiol (E2) and also the unique specificity of affinity for hormones. In this work values of relative binding affinity (RBA) of the UEBP for 57 steroids and their analogs were determined. The affinity of steroids was characterised by the amount of the unlabeled compound needed for 50% inhibition of [3H]-E2 binding with the UEBP. A number of derivatives of estrane and androstane possess an ability to interact with this protein, in contrast to the derivatives of pregnane, stilbene and triphenylethane. Characterized by RBA values, natural steroids are found to have the following order: estriol larger than or equal to E2 greater than 16 alpha-hydroxyestrone = 2 alpha-hydroxytestosterone greater than 16-epiestriol greater than or equal to estetrol greater than or equal to 17-epiestriol greater than or equal to 2-methoxyestradiol greater than or equal to 5 alpha-androstane-3 alpha,17 beta-diol greater than or equal to estrone greater than testosterone greater than or equal to 2 beta-hydroxytestosterone greater than 5 alpha-dihydrotestosterone. Affinity of estrogens and androgens for the UEBP diminishes abruptly after removal of 3- and 17-hydroxy groups, masking of these by ether bonds or changing of 17 beta-hydroxyl to 17 alpha. All the investigated 17 oxo-C19-steroids, 5 beta-derivatives of testosterone, its 6 beta- and 16 alpha-hydroxy metabolites as well as 5 alpha-androstane-3 beta,17 beta-diol and 19-nortestosterone exhibit no essential affinity for the protein. On the basis of the results obtained it is suggested that the binding sites for estrogens and androgens in the UEBP molecule overlap but do not completely coincide.

Androgens↗
PubMed1984

Testosterone metabolism by incubated rat testes after chronic LHRH treatment.

Adult male rats were injected 4 or 8 days with LHRH agonist. After sacrifice the testes were incubated in vitro with or without [4-14C]testosterone. After LHRH-administration the endogenously produced amounts of testosterone and of 7 alpha-hydroxytestosterone, the main testosterone metabolite normally found on incubation of adult rat testes, were drastically reduced when compared with controls. hCG, injected to rats 2 h before sacrifice, increased steroid production. In the LHRH-treated rats, however, the amounts of testosterone and of 7 alpha-hydroxytestosterone produced were much less while an important formation of 5 alpha-androstanediol was observed. The testes of LHRH treated rats metabolized [4-14C]testosterone to a large extent to 5 alpha-reduced and unextractable metabolites while the formation of 7 alpha-hydroxylated metabolites was much reduced. It is concluded that prolonged LHRH treatment provokes not only a depression of the testosterone production but has also an influence on the testicular metabolism pattern of testosterone resulting in a proportionally increased production of 5 alpha-reduced steroids and unextractable metabolites while the formation of 7 alpha-hydroxylated steroids is inhibited.

Androgens↗
PubMed1987

Studies on rat liver microsomal steroid metabolism using 18O-labelled testosterone and progesterone.

In order to investigate the possible involvement of oxygen functions in the rat liver microsomal metabolism of progesterone and testosterone these steroids were specifically labelled with 18O in their oxo-functions and incubated with NADPH supplemented 105,000 g sediments. Gas chromatography-mass spectrometry was used to identify the metabolites formed as well as to quantitate the losses of 18O-label. With 18O-labelled testosterone as substrate two of the major monohydroxylated metabolites, i.e. 2 beta- and 6 beta-hydroxytestosterone were shown to have lost about 25 and 50% of their 18O respectively. A complete retention of label was found in 7 alpha- and 16 alpha-hydroxytestosterone. None of the monohydroxylated progesterone metabolites, i.e. the 2 alpha-, 6 beta- and 16 alpha-hydroxyprogesterone had lost any 18O following incubation with 3,20-18O-labelled progesterone. Control incubation (30', 37 degrees C) with buffer and 18O-labelled progesterone and testosterone revealed no exchange of 18O. Thus the partial loss of 3-18O-label during 2 beta- and 6 beta-hydroxylation of testosterone may indicate a covalent interaction between the steroid 3-oxo-group and one or more cytochrome P-450 species in the rat liver microsomes. In view of the potentiating effect of a 3-imine group in spontaneous 6 beta-hydroxylation the present in vitro data suggest that a steroid protein-interaction may occur via a 3-imine group during 6 beta-hydroxylation of testosterone in rat liver microsomes. Analysis of 5 alpha-reduced metabolites of both progesterone and testosterone showed significant losses of 3-18O, but due to the ease with which 3-oxo-5 alpha-steroids exchange their 3-18O with aqueous media an enzymatically induced loss of 3-18O could not be safely established. The 20-oxido-reductase which converted progesterone did not induce a loss of 20- or 3-18O thus indicating that the oxofunctions were not covalently engaged in the enzymatic binding of the steroid.

Animals↗
PubMed1990

Radioimmunoassay of the anti-cancer agent 4-hydroxyandrostenedione in body fluids.

Antibodies were produced in sheep against a new anti-breast cancer drug 4-hydroxyandrostenedione (4-OHA) using two hapten-ovalbumin conjugates. One of these conjugates (4-hydroxytestosterone-ovalbumin) produced an antiserum suitable for the development of a radioimmunoassay that would allow direct measurement of 4-OHA in plasma at concentrations down to 82 pmol/l, with adequate accuracy, precision and scope for further sensitivity. Although this assay would measure 4-hydroxytestosterone (4-OHT) in addition to 4-OHA, the present data suggest that the magnitude of any interference from endogenous steroids and those derived from 4-OHA could only be minimal. A comparison of solvent-extracted and unextracted samples showed that only unconjugated drug was analysed by this radioimmunoassay. A study of plasma protein binding of 4-OHA showed that at therapeutic concentrations, between 13.5 and 16.5% of plasma 4-OHA was not bound to proteins. This assay system could be a useful adjunct to the future development of 4-OHA as an anti-cancer drug.

Androstenedione↗
PubMed1980

A sensitive enzymeimmunoassay with a fluorimetric end-point for the determination of testosterone in female plasma and saliva.

A fluorimetric enzymeimmunoassay has been developed having the sensitivity (500 fg/assay tube) required for determining testosterone concentrations in female plasma and saliva samples. The assay featured a solid-phase antiserum raised against an 11 alpha-hydroxytestosterone-11-hemisuccinate bovine serum albumin conjugate, an 11 alpha-hydroxytestosterone-11-hemisuccinate horseradish peroxidase conjugate as the "enzyme label", and p-hydroxyphenylacetic acid as the substrate for the development of fluorescence. Specificity was ensured by "extracting" testosterone from samples with a solid-phase anti testosterone-3-/0-carboxymethyl/-oxime serum. The assay was shown to satisfy accepted validation criteria providing results in good agreement with routine radioimmunoassay procedures in both plasma (r greater than 0.98, n=28) and saliva (r greater than 0.99, n=28). In saliva samples collected at 2 hourly intervals by normal healthy women (n=5) testosterone concentrations showed a well defined circadian rhythm: the mean testosterone concentration in early morning samples (174 pmol/litre) fell by 83% in late evening collections. In healthy female volunteers (n=7), mean daily throughout one complete cycle ranged from 50 to 218 pmol/litre. Following dexamethasone administration testosterone concentrations in plasma fell by approximately 50%, and salivary concentrations were undetectable after one hour. This enzymeimmunoassay may be useful in studies of female infertility.

Adult↗
PubMed1994

Synthesis of 11-substituted androstenediones and testosterones as human decidual cell growth inhibitors.

11 alpha-Hydroxytestosterone (1a), 11 beta-hydroxytestosterone (1b), 11 alpha-methoxytestosterone (1c), 11 beta-methoxytestosterone (1d), 11-ketotestosterone (1e), and delta 9(11)-testosterone (1f) were synthesized from hydrocortisone (4b) or 11-epi-hydrocortisone (4a). The six target compounds, together with 11 alpha-methoxyandrostenedione (2c), 11 beta-methoxyandrostenedione, (2d) and their lead compound, testosterone (1), were found to effectively inhibit the growth and differentiation of human decidual cells in culture. There is no observable binding of these compounds to estrogen receptor of rabbit uterus. The introduction of a polar group (e.g., hydroxyl and carbonyl) to C-11 of androstenes decreases both the relative binding affinities to progesterone receptor and the inhibitory effects on human decidual cell growth, while the methylation of 11-hydroxyl group minimizes these effects. The similar effects of a polar group at C-11 of testosterone (1) on the inhibitory effects on human decidual cell growth and the relative binding affinities to progesterone receptor of rabbit uterus may suggest that one of the mechanisms of human decidual cell growth inhibition by these compounds is the anti-progestational activity of these androgens.

Androstenedione↗
PubMed1994

Synthesis of hydroxylated steroid hormones via conjugate addition of a silyl-cuprate reagent.

The synthesis of several hydroxylated steroids via conjugate addition of Fleming's silyl-cuprate reagent, (PhMe2Si)2CuLi, a masked hydroxyl group, to the appropriate enone was studied. By this means 7 alpha-hydroxytestosterone (7) was obtained in good yield from 17 beta-hydroxyandrosta-4,6-dien-3-one (1a), though similar reactions on 17 beta-hydroxyandrosta-1,4-dien-3-one (8) gave a low yield of 1 alpha-hydroxytestosterone (13) chiefly through the poor conversion of the phenylsilyl intermediate into the halogenosilane. 3 beta,16 alpha-Dihydroxy-5 alpha-pregnan-20-one (18b) was obtained in a similar manner from 3 beta-hydroxy-5 alpha-pregn-16-en-20-one and 5 alpha-cholestane-1 alpha,3 alpha-diol(17) was produced from the 1-en-3-one (14) via conjugate addition of the silyl group, reduction of the carbonyl function, and oxidative removal of the silyl group.

Androstenes↗
PubMed1992

Effects of cooked brussels sprouts on cytochrome P-450 profile and phase II enzymes in liver and small intestinal mucosa of the rat.

Male Wistar rats were given semi-synthetic diets supplemented with 0, 2.5, 5 and 20% cooked Brussels sprouts for 2, 7, 14 or 28 days. The effects on several cytochrome P-450 enzymes and phase II enzymes (glutathione S-transferase (GST), glucuronyl transferases 1 and 2 (GT1 and GT2) and DT-diaphorase (DTD)) in the liver and small intestinal mucosa were investigated. From 2 days of exposure onwards Brussels sprouts induced P4501A2 and--to a lesser extent--P4501A1 apoprotein levels in the liver, whereas in the small intestine markedly enhanced P4502B apoprotein levels could be detected. No enhanced P4503A apoprotein levels were observed. The 5 and 20% sprouts diets increased the intestinal pentoxyresorufin depentylation (PROD, 4.5-9-fold), and the hydroxylation of testosterone at the 16 alpha- and 16 beta-site (2.6-4.2-fold) after 2 days of exposure. In addition, the 20% sprouts died also enhanced the intestinal ethoxyresorufin deethylation (EROD) activity (c. 5-fold), the hepatic EROD and PROD activities (c. 2-fold) and the formation of 6 beta-hydroxytestosterone (c. 1.6-fold); the formation of 2 alpha-hydroxytestosterone in the liver was decreased (to c. 70% of the control value). GST activity was induced both in the liver (5 and 20% diet) and intestine (20% diet only) throughout the experiment. The 20% sprouts diet enhanced the hepatic DTD and GT1 activities, whereas the GT2 activity was decreased. The induction of DTD in the small intestine after 2 days (2.5-3.2-fold with 5 and 20% sprouts diets, respectively) diminished during the experiment. These results indicate that dietary exposure to cooked Brussels sprouts for only 2 days can change the metabolic activities of several phase II enzymes and cytochrome P-450 enzymes, of which P4502B is the predominant form induced in the small intestine.

Administration, Oral↗
PubMed1976

Site at which FSH regulates estradiol-17beta biosynthesis in Sertoli cell preparations in culture.

Sertoli cells were isolated from testes of 20-day-old rats and were maintained in primary culture. The ability of these cells to synthesise estradiol-17beta from a variety of exogenous substrates, progesterone, testosterone,androstenedione, 19-hydroxyandrostenedione and 19-hydroxytestosterone in the presence and absence of follicle-stimulating hormone (FSH) was examined. In the presence of each of the substrates alone for 24 h the rate of estradiol-17beta synthesis was very low. FSH (NIH-FSH-S11, 5 mug/ml) stimulated estradiol-17beta synthesis 75-fold when added to medium containing testosterone (5 X 10(-7)M) but caused only marginal stimulation when added to medium containing progesterone (5 X 10(-7) M). Both FSH and dibutyryl cyclic AMP (bu2cAMP) stimulated the conversion of each of the substrates, androstenedione, 19-hydroxyandrostenedione and 19-hydroxytestosterone to estradiol-17beta, and the effects were similar to those observed in the presence of testosterone. These data indicate that, under the culture conditions employed, progesterone is not an effective substrate for conversion to estradiol-17beta by Sertoli cells. Estradiol-17beta synthesis was stimulated by FSH in the presence of the C19 steluences the conversion of androgens to estrogens, either directly or indirectly, at the aromatisation step (i.e. the conversion of 19-hydroxylated androgens to estrogens).

Androstenedione↗
PubMed2001

A study of the expression of the xenobiotic-metabolising cytochrome P450 proteins and of testosterone metabolism in bovine liver.

The expression of xenobiotic-metabolising cytochrome P450 proteins in the liver of cattle was determined using substrate probes and immunologically by Western blot analysis. Compared to the rat, cattle displayed much higher coumarin 7-hydroxylase (CYP2A) and ethoxyresorufin O-deethylase (CYP1) activity but, in contrast, it exhibited much lower debrisoquine 4-hydroxylase (CYP2D) and lauric acid hydroxylase activities (CYP4A). The ethoxyresorufin O-deethylase activity was markedly inhibited by furafylline and a-naphthoflavone, and coumarin 7-hydroxylase by 8-methoxypsoralen. Immunoblot analysis employing antibodies to rat CYP1A1 recognised two immunorelated proteins in bovine liver whose expression appeared to be higher compared with rat. Kinetic studies indicated that a single enzyme is likely to be responsible for the O-deethylation of 7-ethoxyresorufin in bovine liver. When bovine microsomes were probed with antibodies to rat CYP2A2, a single protein was detected in cattle liver. Kinetic analysis followed by construction of Eadie-Hofstee plots indicated that more than one enzyme contributes to the 7-hydroxylation of coumarin. Immunoblot analysis employing antibodies to human CYP2D6 and rat CYP4A1 revealed in both cases a single, poorly expressed immunoreacting band in bovine microsomes. Similar immunoblot studies detected proteins in cattle liver immunorelated to the CYP2B, CYP2C, CYP2E, and CYP3A subfamilies. Bovine microsomes metabolised testosterone but, in contrast to the rat, failed to produce 2alpha- and 16alpha-hydroxytestosterone. On the other hand, bovine microsomes produced levels of another hydroxylated metabolite, possibly 12-hydroxytestosterone. In conclusion, results emanating from this study indicate the presence of proteins in the cattle liver belonging to all the xenobiotic-metabolising families of cytochrome P450.

Androstenedione↗
PubMed1998

Involvement of CYP3A1, 2B1, and 2E1 in C-8 hydroxylation and CYP 1A2 and flavin-containing monooxygenase in N-demethylation of caffeine; identified by using inducer treated rat liver microsomes that are characterized with testosterone metabolic patterns.

Caffeine (CA) is oxidized by rat liver microsomal enzymes to theobromine (TB), paraxanthine (PX), and theophylline (TP) by N-demethylation and to trimethylurate (TMU) by C-8 hydroxylation, In order to identify the specific enzymes responsible for productions of these primary CA metabolites, liver microsomes enriched with various isoforms of cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) are prepared by pretreatment of rats with several inducers. The specific increases in various CYP or FMO activities are identified with the diagnostic testosterone metabolic patterns or the thiobenzamide S-oxidation assay. They are then employed to metabolize the CA. Liver microsomes isolated from rats pretreated with phenobarbital (PB-microsomes) did not have increased FMO activity but had increased activities for hydroxylating the testosterone at 6 beta-(CYP3A1), 16 beta-(CYP2B1), and 2 beta-(CYP3A1) positions. This PB-microsomes had increased activity for TMU production from CA (result of C-8 hydroxylation). Liver microsomes isolated from rats pretreated with acetone (AC-microsomes) had a normal level of FMO activity but had enhanced rates of 6 beta-(CYP3A1) and 2 beta-(CYP3A1) hydroxylations of testosterone. The AC-microsomes again had increased activity for production of TMU. Similarly, the liver microsomes isolated from rats pretreated with dexamethasone (DEX-microsomes) had a normal level of FMO activity but had enhanced rates of forming 6 beta-and 2 beta-hydroxytestosterone (Cyp3A1) as well as androstenedione (CYP3A1). The DEX-microsomes again had increased activity for production of TMU only. Liver microsomes isolated from rats pretreated with 3-methylcholanthrene (MC-microsomes), however, had increased FMO activity and also enhanced rates of forming the 7 alpha-(CYP1A1/2, and 2A1), 6 beta-(CYP3A1), and 2 beta-(CYP3A1) hydroxytestosterone. The MC-microsomes had increased activity for producing all of the four primary metabolites of CA, i.e. the N-demethylation metabolites like TB, PX. and TP, as well as the C-8 hydroxylation metabolite TMU. By the process of association of the obtained results, liver microsomes with increased contents of CYP2B1, 3A1, and 2E1 could catalyze the C-8 hydroxylation at an increased rate producing increased amount of TMU. Increased productions of CA N-demethylation metabolites (TB, PX, and TP) are, however, catalyzed by the increased activities of CYP1A2 and FMO which are associated uniquely with the MC-microsomes.

Animals↗
PubMed1998

Biotransformation XLV. Transformations of 4-ene-3-oxo steroids in Fusarium culmorum culture.

The course of transformations of five 4-ene-3-oxo steroids with varying substituents at C-17 i.e.: 4-androsten-3-one, androstenedione, testosterone, progesterone and 17alpha-hydroxyprogesterone in Fusarium culmorum culture was investigated. All the substrates were hydroxylated either at 12beta and 15alpha, or at 15alpha or 6beta positions, depending on the structure of the substrate. The main product of 4-androsten-3-one transformation was 12beta,15alpha-diol. A similar 12beta,15alpha-diol was obtained from progesterone, but the main product of transformation of this substrate was 15alpha-hydroxyprogesterone. The products of hydroxylation at 6beta or 15alpha positions were isolated from 17alpha-hydroxyprogesterone. The androstenedione and testosterone transformation mixtures contained the same products (6beta-hydroxyandrostenedione, 6beta-hydroxytestosterone, 15alpha-hydroxyandrostenedione and 15alpha-hydroxytestosterone), but the quantities of 6beta- and 15alpha-alcohols varied, depending on the substrate used. During transformations of these two substrates, apart from hydroxylation, ketone-alcohol interconversion at C-17 occurred.

Alcohols↗
PubMed1991

Purification of human placental aromatase cytochrome P-450 with monoclonal antibody and its characterization.

A simple and efficient method is described for the purification of microsomal aromatase cytochrome P-450 from human placenta. The enzyme was solubilized with Emulgen 913 and sodium cholate and subjected to chromatography on a column of Sepharose 4B coupled with a specific monoclonal antibody, followed by hydroxyapatite column chromatography. The specific cytochrome P-450 content of purified aromatase was 13.1 (12-14.8) nmol/mg of protein. Aromatase assays were carried out with reconstituted systems of bovine liver P-450 reductase and dilauroyl-L-alpha-phosphatidylcholine with [1 beta-3H,4-14C]-androstenedione as substrate. The specific activity of purified aromatase was 65.0 (50.6-74.3) nmol.min-1.(mg of protein)-1 or a turnover rate of 5.0 (4.3-5.9) min-1. The total recovery of purified aromatase activity was 32.2%, and P-450 recovery was 17.6%. The Km of immunoaffinity-purified aromatase was 12, 210, 41, and 2830 nM for androstenedione, 16 alpha-hydroxyandrostenedione, testosterone, and 16 alpha-hydroxytestosterone, respectively. The very high Km value for 16 alpha-hydroxytestosterone aromatization gives a reasonable indication that estriol is not the directly aromatized product in the fetoplacental unit of human pregnancy. The aromatase P-450 was subjected to SDS-polyacrylamide gel electrophoresis in increasing quantities. Silver stain detection techniques indicated a single band having a molecular mass of 55 kDa with greater than 97% purity. The stability analysis showed a half-life of over 4 years on storage at -80 degrees C.

Animals↗
PubMed1990

Sequence requirements for cytochromes P450IIA1 and P450IIA2 catalytic activity: evidence for both specific and non-specific substrate binding interactions through use of chimeric cDNAs and cDNA expression.

Cytochrome P450s IIA1 and IIA2, encoded by the CYP2A1 and CYP2A2 genes, display 88% amino acid sequence similarities. The dissimilarities of sequence between these two enzymes are primarily localized within four discrete regions of the polypeptides that are separated by regions of absolute sequence identity. IIA1 specifically hydroxylates the prototype substrate testosterone at the 7 alpha and 6 alpha position with a predominance of 7 alpha metabolite. IIA2, on the other hand, hydroxylates this steroid at eight positions on the molecule, with one of the most abundant metabolites being 15 alpha-hydroxytestosterone. To determine those amino acids responsible for the difference in testosterone hydroxylation specificities, chimeras were constructed between IIA1 and IIA2 cDNAs and expressed in cell culture using vaccinia-virus-mediated cDNA expression. Chimeras, in which the first 355 amino acids correspond to a single enzyme, maintain the specificity associated with that enzyme. Of six chimeras which have substitutions between amino acids 161 and 276, two are inactive and the remaining four give similar metabolite profiles, in which both 7 alpha and 15 alpha hydroxylation specificities have been lost. Two of these four chimeras are diametric apposites, suggesting that modification of either the N-terminal or central regions of the enzymes results in conformational changes that prevent the specific binding interactions responsible for the narrow regioselectivity associated with IIA1 and 15 alpha-hydroxytestosterone formation associated with IIA2.

Cloning, Molecular↗