Probing the active site of reconstituted human placental aromatase [Poster 02]

To gain some insight into the factors that determine active site-ligand binding and to delineate which sites of the androstene nucleus are critical for recognition by the aromatase enzyme, we compared the effect of various substituents located at the biologically important C-2, 6, 11, 16, 17, and 19 positions. Based on two independent measurements, namely optical difference spectra and inhibition of enzyme activity, we report: (a) oxygen functions at positions C-11 do not appear to induce difference spectra although they cause weak inhibition; (b) 2ß- and 16-hydroxyandrostenes induce type-I spectra and weak inhibition; (c) C-6 and C-19-substituted androgens are type-I spectrum inducers and strong inhibitors; (d) the presence of nonspecific binding sites on the aromatase enzyme cannot be ruled out.     

Structure-function studies of aromatase and its inhibitors: a progress report

The utilization of computer modeling, site-directed mutagenesis, inhibition kinetic analysis and reaction metabolite analysis allows us to better understand the structure–function relationship between aromatase and its inhibitors. Our results have helped in determining how steroidal and nonsteriodal aromatase inhibitors bind to the active site of the enzyme. This information has also aided in the understanding of the reaction mechanism of aromatase. Furthermore, our structure–function studies of aromatase have generated important information for predicting how environmental chemicals interact with the enzyme. During the last 2 years, a new aromatase computer model based on the X-ray structure of rabbit cytochrome P450 2C5 has been generated and used to evaluate the results obtained from new aromatase mutants produced in this laboratory. In addition, we have succeeded in the expression and purification of functionally active aromatase using an Escherichia coli expression method. The catalytic properties of this recombinant aromatase are similar to those properties exhibited by the human placental aromatase preparation and the mammalian cell-expressed enzyme. The E. coli expressed aromatase will be very useful for further structure–function studies of aromatase. Our laboratory has also evaluated the growth-inhibiting activity of aromatase inhibitors in estrogen receptor-positive breast cancer using three-dimensional cell cultures of aromatase-over expressing MCF-7 and T-47D cell lines (i.e. MCF-7aro and T-47Daro). Our results demonstrate that these three-dimensional cultures are valuable approaches to assess the growth-inhibiting activity of aromatase inhibitors. Finally, we have identified several phytochemicals to be potent inhibitors of aromatase. To demonstrate the impact of the phytochemicals on estrogen formation in vivo, we showed that the intake of anti-aromatase chemicals from red wine was capable of suppressing MCF-7aro-mediated tumor formation in nude mice and aromatase-induced hyperplasia in a transgenic mouse model in which aromatase is over-expressed in the mammary tissue.     

Growth suppression of MCF-7 human breast cancer cells by aromatase inhibitors: A new system for aromatase inhibitor screening

In our previous study we found that MCF-7 cells possess aromatase activity and stimulate estrogen receptor-mediated growth. The pathways through which androgens are converted to estrogens by aromatase and estrogens interact with estrogen receptors contribute significantly to growth stimulation. The administration of aromatase inhibitor results in suppression of growth stimulation by androgens. This system enabled us to assess directly the biological activities of aromatase inhibitors. Aromatase activity was inhibited in a dose-dependent manner by the addition of aminoglutethimide and CGS 16949A, competitive inhibitors, and of 14-hydroxy-4-androstene-3,6,17-trione and 4-hydroxy-androstenedione, mechanism-based inhibitors. After preincubation with mechanism-based inhibitors, aromatase activity was significantly suppressed, whereas after preincubation with competitive inhibitors, it was adversely increased. These effects were concentration- and time-dependent. Preincubation with competitive inhibitors resulted in augmentation of subsequent androgen stimulation of thymidine incorporation, while preincubation with mechanism-based inhibitors resulted in diminished stimulation by subsequent androgen administration. These results suggest that in MCF-7 cells competitive inhibitors adversely induce aromatase and accelerate the subsequent androgen stimulation of DNA synthesis. Suicide inhibitors are more effective than competitive inhibitors. This system will be useful for aromatase inhibitor screening.     

Expression of human placental aromatase in Saccharomyces cerevisiae

A full-length human placental aromatase cDNA clone, Aro 2, was isolated upon screening a human placental cDNA library with an aromatase cDNA probe and an oligonucleotide probe whose sequence was derived from a human aromatase genomic clone. Nucleotide sequence microheterogeneity was found in the 3'-untranslated region among Aro 2 and in two previously described human aromatase cDNA clones. Both the minor sequence differences and the expression of a single protein species in placental tissue suggest the presence of different alleles for aromatase. Northern blot analyses using one cDNA and two oligonucleotide probes are consistent with the two mRNA messages of 2.9 and 2.5 kilobases arising in human placenta as a consequence of differential processing. Several yeast expression plasmids containing the aromatase cDNA we cloned were constructed. The enzyme was expressed in Saccharomyces cerevisiae. The expressed activity was inhibited by the known aromatase inhibitor, 4-hydroxyandrostenedione. A level of 2 micrograms aromatase/mg partially purified yeast microsomes was estimated by analyses of carbon monoxide difference spectra on microsomal fractions from yeast carrying plasmid pHARK/VGAL. Using [1 beta, 2 beta-3H]androst-4-ene-3,17-dione as the substrate, an apparent Michaels-Menken constant (Km) of 34 nM and a maximum velocity (Vmax) of 23 pmol [3H]water formed per min/mg protein were obtained for the yeast synthesized aromatase by transformation with plasmid pHARK/VGAL. The kinetic results are similar to those determined for human placental aromatase, and suggest that the yeast synthesized aromatase will be useful for further structure-function studies.     

Surgical issues surrounding use of aromatase inhibitors

There are important surgical issues related to the use of the third generation aromatase inhibitors in both the neoadjuvant and adjuvant settings. Neoadjuvant hormone therapy is effective at downstaging tumours, particularly large tumours initially thought to be inoperable or requiring mastectomy. Randomised trials have shown that the newer aromatase inhibitors letrozole and anastrozole increase the numbers of women who are suitable for breast-conservation compared with tamoxifen, and that letrozole is superior to tamoxifen in terms of clinical response.

Aromatase inhibitors are most effective in ER-rich tumours and are clinically and biologically effective in both HER2 positive and negative tumours, whereas HER2 positive tumours show a level of resistance to tamoxifen.

In neoadjuvant studies comparing aromatase inhibitors with tamoxifen, the duration of use has been 3–4 months, by which time any response is usually evident but longer treatment periods produce continued shrinkage and response. The re-excision rate following breast conservation surgery after neoadjuvant hormone therapy is favourable compared with the rates following immediate wide local excision. Local recurrence rates are acceptable in patients undergoing neoadjuvant therapy and breast-conserving surgery providing post-operative radiotherapy is given.

Adjuvant aromatase inhibitors, as well as having an effect on metastatic disease and survival, reduce local and regional recurrence.     

Metabolism of 19-methyl-substituted steroids by human placental aromatase

The 19-methyl analogues of androstenedione and its aromatization intermediates (19-hydroxyandrostenedione and 19-oxoandrostenedione) were evaluated as substrates of microsomal aromatase in order to determine the effect of a 19-alkyl substituent on the enzyme's regiospecificity. Neither the androstenedione analogue [10-ethylestr-4-ene-3,17-dione (1c)] nor the 19-oxoandrostenedione analogue [10-acetylestr-4-ene-3,17-dione (3c)] was converted to estrogens or oxygenated metabolites by placental microsomes. In contrast, both analogues of 19-hydroxyandrostenedione [10-[(1S)-1-hydroxyethyl]estr-4-ene-3,17-dione (2c) and 10-[(1R)-1-hydroxyethyl]estr-4-ene-3,17-dione (2e)] were converted to the intermediate analogue 3c in a process requiring O2 and either NADH or NADPH. No change in enzyme regiospecificity was detected. The absolute configuration of 2e was determined by X-ray crystallography. Experiments with 18O2 established that 3c generated from 2c retained little 18O (less than 3%), while 3c arising from 2e retained a significant amount of 18O (approximately equal to 70%). All four 19-methyl steroids elicited type I difference spectra from placental microsomes in addition to acting as competitive inhibitors of aromatase (KI = 81 nM, 11 microM, 9.9 microM, and 150 nM for 1c, 2c, 2e, and 3c, respectively). Pretreatment of microsomes with 4-hydroxyandrostenedione (a suicide inactivator of aromatase) abolished the metabolism of 2c and 2e to 3c, as well as the type I difference spectrum elicited by 2c and 2e.(ABSTRACT TRUNCATED AT 250 WORDS)     

MR 20492 and MR 20494: two indolizinone derivatives that strongly inhibit human aromatase.

In this study, we describe the synthesis of a new family of indolizinone derivatives designed to fit an extrahydrophobic pocket within the active site of aromatase and to strongly inhibit human aromatase. This could help improve the specificity of the inhibitors. Equine aromatase, very well characterized biochemically, is used as a comparative model. Indeed, in a previous comparison between both human and equine aromatases, we described the importance of the interaction between the inhibitor and this pocket for the indane derivative MR 20814. MR 20492 and MR 20494 are more potent inhibitors of human aromatase (Ki/Km: 1.0+/-0.3 and 0.5+/-0.3, respectively). The Ki/Km for MR 20494 is slightly higher than that obtained for fadrozole (0.1+/-0.0) and Ki/Km for both indolizinone derivatives are lower than those obtained for 4-hydroxyandrostenedione (1.9+/-0.8) and MR 20814 (8.1+/-.7). These new compounds are not enzyme inactivators. Moreover, as indicated by the higher Ki/Km values obtained with equine enzyme (9.0+/-0.6 and 6.1+/-1.6 for MR 20492 and MR 20494, respectively), both human and equine aromatase active sites appear to be structurally different. Difference absorption spectra study (350-500 nm) revealed that MR20492 and MR20494 were characterized by a combination of type-I and -II spectra with both enzymes. This result could be due to the isomerization of the molecule in polar solvent (Z and E forms). The evaluation of these new molecules, as well as 4-hydroxyandrostenedione and fadrozole, on aromatase activity in transfected 293 cell cultures evidenced a strong inhibition (IC50: 0.20+/-0.03 microM, 0.20+/-0.02 microM and 0.50+/-0.40 microM for MR 20494, fadrozole and 4-OHA, respectively) except for MR 20492 (3.9+/-0.9 microM) and MR 20814 (10.5+/-0.6 microM). These results proved that these molecules formed part of a promising family of potent inhibitors and that they penetrate 293 cells, without evidencing any cytotoxicity in Hela cells with MTT assay. This is thus encouraging for the development of new drugs for the treatment of estrogen-dependent cancers, these molecules also constitute new tools for understanding the aromatase active site.     

Effects of aromatase inhibitors on in vitro steroidogenesis by Atlantic salmon (Salmo salar) gonadal and brain tissue

In order to assess the efficacy of selected aromatase inhibitors on Atlantic salmon (Salmo salar) ovarian and brain tissue, in vitro systems were developed for measuring 17beta-estradiol (E(2)) production by these tissues. Isolated vitellogenic follicles, or homogenised whole brains were incubated at 10 degrees C in complete Cortlands solution for 18 or 42 h respectively, and E(2) levels in the medium were determined by RIA. The addition of testosterone to the medium increased E(2) production in all preparations. E(2) production by whole brain homogenate was reduced by co-incubation with the aromatase inhibitors 1,4,6-androstatriene-3,17-dione (ATD), 4-androstene-4-ol-3,17-dione (OHA), aminoglutethimide, fadrozole or miconazole. Fadrozole, ATD, and OHA reduced E(2) production by vitellogenic follicles at a medium concentration of 0.1 microg mL(-1), whereas miconazole was only effective at 10 microg mL(-1). This study demonstrates a simple and rapid screening method for assessing the efficacy of aromatase inhibitors on fish tissues, and that the aromatase inhibitors ATD, OHA and fadrozole are potent inhibitors of both brain and gonadal aromatase in vitro, in Atlantic salmon.     

Aromatase and COX-2 expression in human breast cancers

We have investigated aromatase and the inducible cyclooxygenase COX-2 expression using immunocytochemistry in tumors of a series of patients with advanced breast cancer treated with aromatase inhibitors. Aromatase was expressed in 58/102 breast cancers. This is similar to the percentage previously reported for aromatase activity. Interestingly, aromatase was expressed in a variety of cell types, including tumor, stromal, adipose, and endothelial cells. Since prostaglandin E₂ is known to regulate aromatase gene expression and is the product of COX-2, an enzyme frequently overexpressed in tumors, immunocytochemistry was performed on the tissue sections using a polyclonal antibody to COX-2. Aromatase was strongly correlated (P<0.001) with COX-2 expression. These results suggest that PGE₂ produced by COX-2 in the tumor may be important in stimulating estrogen synthesis in the tumor and surrounding tissue. No correlation was observed between aromatase or COX-2 expression and the response of the patients to aromatase inhibitor treatment. However, only 13 patients responded. Nine of these patients were aromatase positive. Although similar to responses in other studies, this low response rate to second line treatment suggests that tumors of most patients were no longer sensitive to the effects of estrogen. Recent clinical studies suggest that greater responses occur when aromatase inhibitors are used as first line treatment. In the intratumoral aromatase mouse model, expression of aromatase in tumors is highly correlated with increased tumor growth. First line treatment with letrozole was effective in all animals treated and was more effective than tamoxifen in suppressing tumor growth. Letrozole was also effective in tumors failing to respond to tamoxifen, consistent with clinical findings. In addition, the duration of response was significantly longer with the aromatase inhibitor than with tamoxifen, suggesting that aromatase inhibitors may offer better control of tumor growth than this antiestrogen.     

Aromatase inhibitors and inactivators in the treatment of advanced breast cancer

Advanced breast cancer remains incurable. For these patients, durable response and minimal toxicity are the main goals of current therapy. The antiestrogen tamoxifen has proved to be a significant advance in the treatment of breast cancer. Due to its partial estrogen activity, long term medication with tamoxifen has been found to cause endometrium proliferation wich can result in cancer in some patients. Reduction of estrogen production identified the aromatase inhibitors. Both steroidal substrate analog, type I inactivator, wich inactivate the enzyme and non-steroidal competitive reversible, type II inhibitors, are now avaiable. Two new 3(rd) generation aromatase inactivators have recently completed phase III evaluation (anastrozole and letrozole) and we have some results investigating one of the new 3(rd) generation aromatase inhibitors (exemestane). The 3(rd) generation aromatase inhibitors and inactivators are better tolerated and more effective than each of our current standard 2(nd) line endocrin therapies. These agents are being directly compared with standard adjuvant medication, tamoxifen, or are being evaluated in different sequences.     

Biochemical and computational insights into the anti-aromatase activity of natural catechol estrogens

High levels of endogenous estrogens are associated with increased risks of breast cancer. Estrogen levels are mainly increased by the activity of the aromatase enzyme and reduced by oxidative/conjugative metabolic pathways. In this paper, we demonstrate for the first time that catechol estrogen metabolites are potent aromatase inhibitors, thus establishing a link between aromatase activity and the processes involved in estrogen metabolism. In particular, the anti-aromatase activity of a set of natural hydroxyl and methoxyl estrogen metabolites was investigated using biochemical methods and subsequently compared with the anti-aromatase potency of estradiol and two reference aromatase inhibitors. Catechol estrogens proved to be strong inhibitors with an anti-aromatase potency two orders of magnitude higher than estradiol. A competitive inhibition mechanism was found for the most potent molecule, 2-hydroxyestradiol (2-OHE(2)) and a rational model identifying the interaction determinants of the metabolites with the enzyme is proposed based on ab initio quantum-mechanical calculations. A strong relationship between activity and electrostatic properties was found for catechol estrogens. Moreover, our results suggest that natural catechol estrogens may be involved in the control mechanisms of estrogen production.     

A delayed gonadotropin-dependent and growth factor-mediated activation of the extracellular signal-regulated kinase 1/2 cascade negatively regulates aromatase expression in granulosa cells

Human chorionic gonadotropin and human FSH (hFSH) elicit a transient increase in ERK1/2 phosphorylation lasting less than 60 min in immature granulosa cells expressing a low density of gonadotropin receptors. In cells expressing a high density of receptors, human chorionic gonadotropin and human FSH elicit this fast transient increase in ERK1/2 phosphorylation and also a delayed and more sustained increase that is detectable after 6-9 h. Both the early and delayed increases in ERK1/2 phosphorylation can be blocked with inhibitors of protein kinase A, the epidermal growth factor receptor kinase, metalloproteases, and MAPK kinase. The delayed effect, but not the early effect, can also be blocked with an inhibitor of protein kinase C. Because the delayed increase in ERK1/2 phosphorylation correlates with low aromatase expression in response to gonadotropins, we tested the effects of these inhibitors on aromatase expression. These inhibitors had little or no effect on gonadotropin-induced aromatase expression in cells expressing a low density of receptors, but they enhanced gonadotropin-induced aromatase expression in cells expressing a high density of receptors. Phorbol esters also induced a prolonged increase in ERK1/2 phosphorylation and, when added together with hFSH, blocked the induction of aromatase expression by hFSH in cells expressing a low density of hFSH receptor. A MAPK kinase inhibitor reversed the inhibitory effect of the phorbol ester on aromatase induction. We conclude that the effects of gonadotropins on ERK1/2 phosphorylation are mediated by epidermal growth factor-like growth factors and that the delayed effect is partially mediated by protein kinase C and acts as a negative regulator of aromatase expression.     

Intracellular aromatase and its relevance to the pharmacological efficacy of aromatase inhibitors

An important feature of the pharmacological profile of aromatase inhibitors is the ability of the various inhibitors to inhibit intracellular aromatase. It is now well documented that a large proportion of breast tumors express their own aromatase. This intratumoral aromatase produces estrogen in situ and therefore may contribute significantly to the amount of estrogen to which the cell is exposed. Thus it is not only important that aromatase inhibitors potently inhibit the peripheral production of estrogen and eliminate the external supply of estrogen to the tumor cell, but that they in addition potently inhibit intratumoral aromatase and prevent the tumor cell from making its own estrogen within the cell. To study the inhibition of intracellular aromatase we have compared the aromatase-inhibiting potency of the non-steroidal aromatase inhibitors, letrozole, anastrozole and fadrozole in a variety of model cellular endocrine and tumor systems which contain aromatase. We have used hamsters ovarian tissue fragments, adipose tissue fibroblasts from normal human breast, the MCF-7Ca human breast cancer cell line transfected with the human aromatase gene and the JEG-3 human choriocarcinoma cell line. Although letrozole and anastrozole are approximately equipotent in a cell-free aromatase system (human placental microsomes), letrozole is consistently 10–30 times more potent than anastrozole in inhibiting intracellular aromatase in intact rodent cells, normal human adipose fibroblasts and human cancer cell lines. Whether these differences between letrozole and anastrozole are seen in the clinical setting will have to await the results of clinical trials which are currently in progress.     

Inhibition of aromatase: insights from recent studies

Aromatase is the rate limiting enzyme that catalyzes the conversion of androgens to estrogens. Blockade of this step allows treatment of diseases that are dependent upon estrogen. Over the past two decades, highly potent and specific aromatase inhibitors have been developed which block total body aromatization by over 99%. An important recent question is whether aromatase inhibitors are superior to the antiestrogens for treatment of hormone-dependent breast cancer. The third generation aromatase inhibitors have been compared to tamoxifen for the treatment of breast cancer in the advanced, adjuvant, and neoadjuvant settings. All of these studies suggest the superiority of aromatase inhibitors over tamoxifen. The mechanism responsible for the superiority of the aromatase inhibitors relates to the estrogen agonistic effects of tamoxifen. During exposure to estrogen deprived conditions and to tamoxifen, breast cancer cells adapt and upregulate the MAP kinase and PI-3 kinase pathways. These growth factor signaling pathways potentiate the estrogen agonistic properties of tamoxifen. Data from a large adjuvant therapy trial (ATAC trial) provide evidence that the aromatase inhibitors may also be superior for breast cancer prevention. The mechanism for superiority in this setting probably relates to the genotoxic effects of estradiol metabolites. The aromatase inhibitors may be also useful for the treatment of endometriosis and for ovulation induction as evidenced by preliminary data. The recent advances in development of the aromatase inhibitors clearly demonstrate the utility of these agents for treatment of breast cancer and potentially for other indications.     

Aromatase inhibitors: assessment of biochemical efficacy measured by total body aromatase inhibition and tissue estrogen suppression

The implementation of aromatase inhibitors for treatment of early and metastatic breast cancer has been one of the major improvements in endocrine therapy of breast cancer. Measurement of endocrine effects of aromatase inhibition in vivo has been a major tool in the process of evaluating novel compounds. Biochemical efficacy of aromatase inhibitors in vivo may be determined from their effects on “total body aromatization” as well changes in plasma and tissue estrogen levels. Due to high sensitivity, tracer methods allowing calculation of whole body aromatase inhibition are still considered the gold standard. The method developed by our group in collaboration with the Royal Marsden Hospital and the results of this joint program are summarized and discussed. These studies allowed classification of the different aromatase inhibitors and their optimal dosage, selecting the best compounds for clinical evaluation. In vivo total body aromatase assessment is a work-consuming method, allowing such studies to be conducted in a limited number of patients only. In contrast, plasma estrogen measurement is a cruder but simpler method, allowing screening of larger groups of patients. As plasma estrogens arise through passive diffusion of estrogens synthesized in different body compartments, plasma estrogens, as well as total body aromatase assessment, present a rough estimate of total body tissue estrogen production, and changes associated with treatment with aromatase inhibitors reflect the effects on tissue estrogen production in general. However, plasma estrogen levels do not correlate to breast cancer tissue estrogen levels. This is due to the endocrine autonomy of breast cancer tissue with significant local estrogen production in some tumors. Thus, direct measurement of intratumor estrogens is demanded to evaluate the effects of aromatase inhibitors in malignant target tissues. Our group has developed a highly sensitive HPLC-RIA for the simultaneous measurement of estrone, estradiol, and estrone sulfate in malignant breast tissue samples, and we are currently using this method to assess alterations in intratumor estrogen levels during treatment with different aromatase inhibitors.     

Structure-activity relationships of 3-deoxy androgens as aromatase inhibitors. Synthesis and biochemical studies of 4-substituted 4-ene and 5-ene steroids

As part of our investigation into the structure-activity relationship of a novel class of aromatase inhibitors, two series of 3-deoxy androgens, androst-5-en-17-ones with a non-polar alkoxy (5 and 6), alkyl (20-22), or phenylalkyl (23 and 24) group at C-4beta and 4-acyloxyandrost-4-en-17-ones (29-32, and 34) were synthesized and evaluated. The 4beta-alkyl and 4beta-phenylalkyl compounds were obtained through reaction of 4alpha,5alpha-epoxy steroid (8) with RMgBr (R: alkyl and phenylalkyl) followed by dehydration of the 4beta-substituted 5alpha-hydroxy products (15-19) with SOCl(2) as key reactions. Acylation of 4alpha,5alpha-diol (25) with (RCO)(2)O in pyridine and subsequent dehydration with SOCl(2) gave the 4-acyloxy steroids. All of the steroids studied, except for 4-acetoxy-19-ol (34) that was a non-competitive inhibitor of human placental aromatase, blocked aromatase activity in a competitive manner. 4-Benzoyloxy- and 4-acetoxy steroids (31) and (32) were the most powerful inhibitors of aromatase (K(i)=70 and 60nM, respectively). Elongation of an acetoxy group in a series of 4-acyloxy steroids or a methyl group in a series of 4beta-alkyl steroids decreased affinity for aromatase principally in relation to carbon number of the acyl or alkyl function. The present findings are potentially useful for understanding the spatial and electronic nature of the binding site of aromatase as well as for developing effective aromatase inhibitors.     

Postmenopausal estrogen synthesis and metabolism: alterations caused by aromatase inhibitors used for the treatment of breast cancer

Inhibition of postmenopausal estrogen production by aromatase inhibitors is an established drug treatment modality for postmenopausal breast cancer. In this article postmenopausal estrogen disposition and the alterations caused by treatment with aromatase inhibitors are reviewed. Recent investigations have challenged the hypothesis that aromatization of androstenedione into estrone is the sole production pathway for estrogens in postmenopausal women. The finding that estrogens persist in the plasma of patients receiving aminoglutethimide treatment despite a near total inhibition of the aromatase enzyme suggests that alternative pathways for estrogen synthesis exist. While nonspecific actions of aromatase inhibitors may be disadvantageous, certain effects may also be beneficial. Recent findings that aminoglutethimide may induce estrone sulfate metabolism questions whether this "prototype" aromatase inhibitor might have a dual mechanism of action. The importance of investigating the possible influence of different aromatase inhibitors on all components of estrogen disposition is considered.     

Immunoaffinity purification of aromatase cytochrome P-450 from human placental microsomes, metabolic switching from aromatization to 1 beta and 2 beta-monohydroxylation, and recognition of aromatase isozymes

Microsomal estrogen synthetase (aromatase) cytochrome P-450 was purified from fresh human placental microsomes by monoclonal anti-aromatase P-450 antibody-Sepharose 4B chromatography. The purified P-450 showed a single band of 55 kDa on SDS-polyacrylamide gel electrophoresis and the aromatase specific activity on reconstitution was 70 nmol/min/mg protein. The purified P-450 was stable with a t 1/2 of approximately 2 years on storage at -90 degrees C and showed Km = 43 nM for androstenedione aromatization. However, it was unstable under spectral measurement conditions in the presence of sodium dithionite and carbon monoxide and the carbon monoxide difference spectra showed a maximum at 450 nm and a specific content of 9.1 nmol of P-450/mg protein, giving a turnover number of approximately 7.7 per min for the purified aromatase. The one-step immunochemical purification method gave a 490-fold increase of specific activity with 55% yield of aromatase activity of the original microsomes. Analysis of androgen metabolism by the purified aromatase and an apparent large kinetic isotope effect found at the secondary positions when using [19(-3)H3, 4(-14)C] androgens revealed metabolic switching from the first 19-hydroxylation to 1 beta- and 2 beta- monohydroxylation by aromatase. Substrate specificity for [19(-3)H3]androstenedione and testosterone was indicated by differences in the extent of metabolic switching (18% and 30%) and in the 2 beta/1 beta ratio (60/40 and 10/90, respectively). The mouse monoclonal antibody used for immunoaffinity purification suppresses aromatase activity of human placenta, but was totally ineffective for aromatase in goldfish brain and rat ovary. Rabbit polyclonal antibodies to human placental aromatase P-450 suppressed both human placental and rat ovarian aromatase but were ineffective for goldfish brain aromatase. The study indicates that they are isozymes of aromatase based on different structures of P-450.