Human aromatase: perspectives in biochemistry and biotechnology.

Aromatase (CYP19) is involved in steroidogenesis, catalyzing the conversion of androgens into estrogens through a unique reaction that causes the aromatization of the A ring of the steroid. The enzyme is widely distributed and well conserved among species as it plays a crucial role in physiological processes such as control of reproduction and neuroprotection. It has also been a subject of intense research both at the biotechnological level in drug development due to its involvement in estrogen-dependent tumors and at a fundamental biochemical level because there are numerous questions regarding its reaction mechanism. This review will report the great progress made in this area.

History of Estrogen: Its Purification, Structure, Synthesis, Biologic Actions, and Clinical Implications.

This mini-review summarizes key points from the Clark Sawin Memorial Lecture on the History of Estrogen delivered at Endo 2018 and focuses on the rationales and motivation leading to various discoveries and their clinical applications. During the classical period of antiquity, incisive clinical observations uncovered important findings; however, extensive anatomical dissections to solidify proof were generally lacking. Initiation of the experimental approach followed later, influenced by Claude Bernard's treatise "An Introduction to the Study of Experimental Medicine." With this approach, investigators began to explore the function of the ovaries and their "internal secretions" and, after intensive investigations for several years, purified various estrogens. Clinical therapies for hot flashes, osteoporosis, and dysmenorrhea were quickly developed and, later, methods of hormonal contraception. Sophisticated biochemical methods revealed the mechanisms of estrogen synthesis through the enzyme aromatase and, after discovery of the estrogen receptors, their specific biologic actions. Molecular techniques facilitated understanding of the specific transcriptional and translational events requiring estrogen. This body of knowledge led to methods to prevent and treat hormone-dependent neoplasms as well as a variety of other estrogen-related conditions. More recently, the role of estrogen in men was uncovered by prismatic examples of estrogen deficiency in male patients and by knockout of the estrogen receptor and aromatase in animals. As studies became more extensive, the effects of estrogen on nearly every organ were described. We conclude that the history of estrogen illustrates the role of intellectual reasoning, motivation, and serendipity in advancing knowledge about this important sex steroid.

Multiple forms of aromatase and response of breast cancer aromatase to antiplacental aromatase II antibodies.

Two distinct aromatase-active protein complexes are solubilized by use of deoxycholate and separated by diethylamino-ethyl-cellulose chromatography from lyophilized powder of 900 X g precipitate fraction of human term placenta. Aromatase activity to produce estriol, the major estrogen of human pregnancy, was designated to be aromatase I activity and measured by estriol formation from 16 alpha-hydroxytestosterone. Aromatases II activity was the designation for that which produces estrone plus estradiol and was measured by androstenedione aromatization. Aromatases II and I are eluted with 0.25 M and 0.5 M Tris buffer, respectively, from diethylaminoethyl-cellulose column in an Mr 2 million soluble complex. Each has a minimum active Mr 135,000 subunit, which is isolated by Bio-Gel filtration in the presence of detergents, and consists of a reduced nicotinamide adenine dinucleotide phosphate:cytochrome P-450 reductase (Mr 83,000) and a cytochrome P-450 (Mr 52,000). Aromatase II was found to be the major aromatase, containing approximately five times more aromatase activity, reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase activity, cytochrome P-450, and protein than did aromatase I. Antibodies raised in rabbits against aromatase II and its reductase suppressed aromatase II activity of breast cancer tissues, as well as of adult male lung tissue, placental microsomes, and solubilized aromatase. The breast carcinoma specimens responded to the antibodies in different degrees, but there was no response to antibodies against rat liver cytochrome P-450. The results indicate similar antigenic structures for breast cancer and placental aromatase but not for rat liver cytochrome P-450.

Molecular characterization and expression of equine testicular cytochrome P450 aromatase.

We characterized testicular equine aromatase and its expression. A 2707 bp cDNA was isolated, it encoded a polypeptide of 503 residues with a deduced molecular mass of 57.8 kDa. The sequence features were those of a cytochrome P450 aromatase, with a 78% polypeptide identity with the human counterpart. The gene has a minimal length of 74 kb comprising at least 9 exons and expresses a 2.8 kb mRNA in the testis. Transient cDNA transfections in E293 cells and in vitro translations in a reticulocyte lysate system allowed aromatase protein and activity detections. The activity increased with androstenedione as substrate in a dose-dependent manner. The isolation of testicular aromatase by a new immunoaffinity method demonstrated that the protein could exist either glycosylated or not with a 2 kDa difference. All these results taken together allow new structural studies to progress in the understanding of this cytochrome P450.

Regulation by follicle-stimulating hormone of the synthesis of aromatase cytochrome P-450 in human granulosa cells.

The effects of FSH to increase the activity of aromatase, as well as the synthesis of the components of the aromatase enzyme complex, have been studied in human ovarian granulosa cells obtained from women undergoing oocyte retrieval. FSH increased aromatase activity, as well as the synthesis of aromatase cytochrome P-450 (P-450AROM) in a time-dependent fashion, whereas in the absence of FSH, both activity and synthesis declined with duration of culture. The effect of FSH was mimicked by forskolin, an activator of adenylate cyclase. FSH also increased the synthesis of NADPH-cytochrome P-450 reductase, but to a relatively modest extent. The levels of hybridizable mRNA species encoding cytochrome P-450AROM of lengths 3.0, 2.4, and 1.6 kilobases were also increased with FSH treatment. It is concluded that the regulation of aromatase activity by FSH in human granulosa cells is mediated primarily by changes in the synthesis of cytochrome P-450AROM, that this action of FSH is mediated by cAMP, and that the changes in cytochrome P-450AROM synthesis are the consequences of changes in the levels of mRNA encoding this enzyme.

Structure of an activity suppressing Fab fragment to cytochrome P450 aromatase: insights into the antibody-antigen interactions.

The crystal structure of a Fab fragment (Fab3-2C2) of a monoclonal antibody raised against aromatase cytochrome P450 P450arom) has been determined at 3.0 A resolution. P450arom is a membrane bound enzyme responsible for the catalysis of indrogens to estrogens, the process of aromatization, and hence has been implicated in hormone-dependent breast cancer. The Fab fragment of MAb3-2C2 IgG suppresses P450arom activity in a dose dependent manner. The Fab3-2C2 molecule crystallizes n the space group P2(1)2(1)2(1) with a unit cell of a= 154.89 A, b = 73.51 A, and c= 36.90 A. The crystal structure consists of a light and a heavy chain in the asymmetric unit, each characterized by the greek-key antiparallel beta barrel folding seen in all Fab structures. The average elbow angle between the two domains is 143 degrees. Modeling of the interactions between the variable domains of the antibody and a known model of P450arom maps the epitope to a region of the enzyme that is consistent with the available biochemical data and the activity-suppressing function of the antibody. The epitope mapping result is further supported by the inability of MAb3-2C2 IgG to suppress the activity of, or to interact with placental porcine P450arom, which is 81% identical (86% similar) to human P450arom but has a few key substitutions in the putative epitope region.

The intracellular control of aromatase activity by 5 alpha-reduced androgens in human breast carcinoma cells in culture.

Four cell lines, each derived from a primary tumor from a patient with breast carcinoma, were grown to confluence in alpha-Minimum Essential Medium with 15% fetal calf serum and incubated for 24 h with [3H]androstenedione. The two lines (SA and PP) with the lowest formation of estrone and estradiol (less than 0.1% conversion) were the most active in the formation of the 5 alpha-reduced androgen metabolites androsterone (AND), 5 alpha-androstanedione (5 alpha-A-dione), and dihydrotestosterone (DHT). The two lines with the highest aromatase activity (DM and MD) had the lowest formation of 5 alpha-reduced metabolites. To determine if the 5 alpha-reduced androgen metabolites formed within the breast carcinoma cells could influence aromatase activity, the MD line was further studied. After 24-h preincubation with AND, DHT, or 5 alpha-A-dione at concentrations of 10(-6), 10(-7), and 10(-8) M, [3H]androstenedione was added to the culture medium, and aliquots were removed at 0, 4, 8, and 24 h. An 8-h incubation period was found to be optimum for inhibition studies. In comparison to control levels of estrone (2.5%) and estradiol (0.35%) formation, inhibition of aromatization was evident with all three compounds at 10(-8) M, with 5 alpha-A-dione producing the greatest inhibition (50%). At 10(-7) M, inhibition ranged from 45% (AND) to 70% (5 alpha-A-dione), and at 10(-6) M, inhibition was greater than 90% for each compound. 5 alpha-A-dione produced slightly greater inhibition than AND or DHT at each concentration tested. Since each of these compounds was capable of inhibiting aromatization, the cumulative effect of these 5 alpha-reduced metabolites could be an important factor in the intracellular regulation of aromatase activity.

Structure-activity relationships of a new family of steroidal aromatase inhibitors. 1. Synthesis and evaluation of a series of analogs related to 19-[(methylthio)methyl]androstenedione (RU54115).

During the course of a study aimed at the search for new potent aromatase inhibitors, several new androstenedione analogs were synthesized and evaluated. This study led to the discovery of 19-[(methylthio)methyl]androsta-4,9(11)-diene-3,17-dione (7; RU54115) already described by our laboratory. The object of the present series of papers is to disclose the result of the structure-activity relationship studies that gave rise to this compound. This first part deals mainly with the substitution in the 19-position of the steroid nucleus. Several parameters were varied, the length of the chain and its rigidity and branching, as well as the nature of the heteroatom itself and its substitution. The interaction of these new compounds with human placental aromatase in competition with the substrate androstenedione was studied by difference visible spectroscopy. The in vivo aromatase-inhibiting activities were evaluated by measuring the estradiol lowering after oral administration of the compounds to PMSG-primed female rats.

Expression and purification of a recombinant form of human aromatase from Escherichia coli.

Aromatase converts androgen to estrogen, a hormone that plays an important role in the development of breast cancer. Aromatase inhibitors have been shown to be a useful endocrine regimen for estrogen-dependent breast cancer. Structure-function studies of aromatase can generate critical structural information for designing highly potent and specific inhibitors. However, aromatase structure-function studies have been hampered by a lack of purified protein. In this report, we describe the construction and expression of a recombinant derivative of human aromatase in Escherichia coli using the pET vector system, and the purification of the enzyme by means of nickel-agarose affinity chromatography. We examined the expression of the full-length, Del-38, C-6xHis-tagged Del-38, and NC-6xHis-tagged Del-38 forms of aromatase. The recombinant aromatase without the first 38 amino acids from the amino-terminus (i.e. Del-38) was found to have a higher activity than the full-length enzyme. Moreover, the addition of two separate hexameric histidine tags at both the amino and the carboxyl-termini (i.e. NC-6xHis-tagged Del-38) increased the binding affinity of the recombinant enzyme to the nickel-agarose. The expressed aromatase (i.e. NC-6xHis-tagged Del-38 aromatase) was eluted from the nickel-agarose with 80 mM EDTA. The total aromatase activity of the 80 mM EDTA-eluted fractions was significantly higher than the detergent-solubilized protein extract, indicating a renaturation process during the nickel-agarose affinity chromatography. Purified aromatase exhibited a single band when analyzed by SDS-PAGE, and activity up to 5.8 nmol/mg/min was obtained using the tritiated water release assay. The K(m) value for androstenedione was determined to be 62+/-24 nM by enzyme kinetic analysis. The recombinant aromatase preparation was also characterized by reduced CO-difference spectral analysis, reaction product extraction assay, and inhibition studies using two aromatase inhibitors (letrozole and anastrozole). The results indicate that the recombinant aromatase from E. coli has catalytic properties identical to those of the enzyme expressed in human tissue and will be very useful for further structure-function studies of aromatase.

Expression of a recombinant derivative of human aromatase P450 in insect cells utilizing the baculovirus vector system.

Aromatase P450 (P450arom) is the enzyme responsible for estrogen biosynthesis. Studies of the relationship of the function of this enzyme to its structure have been hampered by lack of a suitable preparation. In the present report we describe the expression of a recombinant derivative of P450arom in insect cells by means of the baculovirus vector system. This protein, which lacks the first 41 amino acids from the N-terminus, and hence the membrane-spanning region, has spectral properties and activity similar to that of the wildtype protein. Moreover, the presence of a hexameric histidine tag at the C-terminus permits its facile purification by means of nickel-agarose affinity chromatography. This system permits the synthesis of quantities of a biologically active derivative of P450arom suitable for studies designed to explore the relationship of function to structure.

Novel properties of human placental aromatase as cytochrome P-450: purification and characterization of a unique form of aromatase.

Aromatase has been purified to homogeneity from human placental microsomes based on detection of its catalytic activities in the eluates from columns of octylamino-Sepharose 4B, hydroxylapatite, Mono S, hydroxylapatite HCA, and Mono Q. The purified preparation shows only one band corresponding to the apparent subunit molecular weight of 51,000 daltons on sodium dodecyl sulfate-polyacrylamide gel. The aromatase in the presence of NADPH and NADPH-cytochrome P-450 reductase converts testosterone to 17 beta-estradiol with the high specific activity of 103 nmol/min/mg of protein. However, whether the preparation is reduced by sodium dithionate chemically or by NADPH and the reductase enzymatically, its reduced, CO-difference spectrum has no peak at about 450 nm and has only a small peak at about 420 nm, probably due to its inactivation in spite of the catalytically full activity in the same preparation. The absolute spectrum of the aromatase exhibits a Soret peak at 423 nm in the absence of testosterone and addition of testosterone to the aromatase sample makes its absorption peak shift gradually from 423 to 393 nm (high spin type peak), which is a usual characteristic in the spectrum of cytochrome P-450. The reconstituted aromatase system efficiently catalyzes aromatization of 4-androstenedione, 19-hydroxy-4-androstenedione as well as testosterone. 16 alpha-Hydroxy-4-androstenedione and 16 alpha-hydroxytestosterone are also aromatized less efficiently and 19-nortestosterone is aromatized least efficiently. The reconstituted aromatase could scarcely oxidize various xenobiotics examined, suggesting a strict and narrow substrate specificity of this enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Steroidal inhibitors as chemical probes of the active site of aromatase.

Androstenedione analogs containing 7 alpha-substituents have proven to be potent inhibitors of aromatase in human placental microsomes, in MCF-7 mammary cell cultures, and in JAr choriocarcinoma cells. Recent investigations have focused on the use of mechanism-based inhibitors, such as 7 alpha-substituted 1,4-androstadienediones, to biochemically probe the active site of aromatase. Inhibition kinetics were determined under initial velocity conditions using purified human placental cytochrome P450arom protein in a reconstituted system. Derivatives of 1,4-androstadiene-3,17-dione and 1,4,6-androstatriene-3,17-dione exhibited high affinity in the purified enzyme system. 7 alpha-(4'-Amino)phenylthio-1,4-androstadiene-3,17-dione, abbreviated 7 alpha-APTADD, demonstrated rapid time-dependent, first-order inactivation of reconstituted aromatase activity only in the presence of NADPH. The apparent Kinact for 7 alpha-APTADD is 11.8 nM, the first-order rate of inactivation is 2.72 x 10(-3) sec-1, and the half-time of inactivation at infinite inhibitor concentration is 4.25 min. The values for the rate constant and half-time of inactivation are similar to those observed in the placental microsomal assay system. Further studies were performed with radioiodinated 7 alpha-(4'-iodo)phenylthio-1,4-androstadienedione, 7 alpha-IPTADD, and the reconstituted aromatase system. Incubations with [125I] 7 alpha-IPTADD were followed by protein precipitation, solvent extraction, and column chromatography. Analysis of the isolated cytochrome P450arom by gel electrophoresis and autoradiography demonstrated the presence of only one radioactive band, which corresponded to the protein staining band for cytochrome P450arom. HPLC radiochromatographic analysis of the isolated cytochrome P450aroM confirmed the presence of only one radioactive peak coeluting with the u.v. peak for cytochrome P450arom. Peptide mapping analysis by reverse-phase HPLC of digested inhibitor-cytochrome P450arom complex demonstrates that the radioactive inhibitor is covalently bound to a lipophilic fragment. In summary, these inhibitors produced enzyme-catalyzed inactivation of reconstituted aromatase activity, and radioiodinated 7 alpha-IP-TADD binds covalently to the cytochrome P450arom.

Biological characterization of A-ring steroids.

Hydroxylated 2,19-methylene-bridged androstenediones were designed as potential mimics of enzyme oxidized intermediates of androstenedione. These compounds exhibited competitive inhibition with low micromolar affinities for aromatase. These inhibitory constants (Ki values) were 10 times greater than the 2,19-methylene-bridged androstenedione constant (Ki = 35-70 nM). However, expansion of the 2,19-carbon bridge to ethylene increased aromatase affinity by 10-fold (Ki = 2 nM). Substitution of a methylene group with oxygen and sulfur in this expanded bridge resulted in Ki values of 7 and 20 nM, respectively. When the substituent was an NH group, the apparent inhibitory kinetics changed from competitive to uncompetitive. All of these analogs exhibited time-dependent inhibition of aromatase activity following preincubation of the inhibitor with human placental microsomes prior to measuring residual enzyme activity. Part of this inhibition was NADPH cofactor-dependent for the 2,19-methyleneoxy- but not for the 2,19-ethylene-bridged androstenedione. The time-dependent inhibition for these four analogs was very rapid since they exhibited tau 50 values, the t1/2 for enzyme inhibition at infinite inhibitor concentration, of 1 to 3 min. These A-ring-bridged androstenedione analogs represent a novel series of potent steroidal aromatase inhibitors. The restrained A-ring bridge containing CH2, O, S, or NH could effectively coordinate with the heme of the P450 aromatase to allow the tight-binding affinities reflected by their nanomolar Ki values.

Studies on the catalytic function of aromatase: aromatization of 6-alkoxy-substituted androgens.

To gain insight into the catalytic function of aromatase, we studied aromatization of a series of 6alpha- and 6beta-ether-substituted (methoxy, ethoxy, and n-butoxy) androst-4-ene-3,17-dione (AD) steroids (1 and 2) and their androsta-1,4-diene-3,17-dione (ADD) derivatives (3 and 4) with human placental aromatase by gas chromatography-mass spectrometry (GC-MS). Among the steroids examined, 6beta-methoxy and 6beta-ethoxyADDs (4a and 4b) are suicide substrates of aromatase. All of the steroids were found to be converted into the corresponding 6-alkoxy estrogens. Introduction of the alkoxy groups at C-6 of AD or ADD decreased the ability of these to serve as a substrate of aromatase. In 6alpha-alkoxy steroid series, compounds 1 and 3, the aromatization rate increased by elongating the 6-methoxy group up to the n-butoxy group whereas, in the 6beta-isomers series, 2 and 4, the rate decreased due to this structural modification. 6beta-Alkoxy steroids, 2 and 4, including the suicide substrates, were extremely poor substrates for the aromatization reaction. Apparent K(m) values obtained for 6alpha-alkoxy compounds 1 and 3 were similar to each other, ranging from 92 to 111nM, as shown by their previously-obtained K(i) values. The findings indicate that the stereochemistry as well as the bulkiness of the 6-ether-substituent play an important role in the ability to serve as a substrate. It is also predicted that the aromatization reaction and the mechanism-based inactivation reaction would be related and have a definite partition number which is characteristic to the compound in a series of suicide substrates.

European sea bass (Dicentrarchus labrax L.) cytochrome P450arom: cDNA cloning, expression and genomic organization.

Cytochrome P450arom, a key enzyme in the hormonal steroidogenic pathway, mediates the conversion of androgens to estrogens. This work describes the molecular cloning of the cDNA encoding the European sea bass (Dicentrarchus labrax L.) cytochrome P450arom by means of reverse transcriptase and polymerase chain reaction (RT-PCR) and 5' and 3'-rapid amplification of cDNA ends (RACE) analyses. The cDNA is 1822bp in length and encodes a putative protein of 517 amino acids. Northern blot analysis revealed that the ovary expressed a transcript of about 2.2kb in size. Analysis of the deduced amino acid sequence indicated 62-86% identity with ovarian P450arom of other teleost fish, the highest identity being found with the Japanese flounder, Paralichthys olivaceous. Identity was lower (56-65%) with the P450arom forms first reported in teleost brain. Only 52% identity was observed with the corresponding fragment of the cartilaginous fish, Dasyatis sabina. RT-PCR revealed that the sea bass P450arom mRNA was also expressed, at low levels, in testis and brain. Between the 5' and 3'-untranslated terminal regions (UTR), the sea bass CYP19 gene contains eight introns. All introns conform to the GT/AG rule for RNA splicing and are inserted in exactly the same positions as those found in Oryzias latipes and the human CYP19 gene.

Endogenous steroid hormones and local aromatase activity in the breast.

To test the hypothesis of an increased activity of the enzyme aromatase in adipose tissue from affected when compared with non-affected quadrants of patients with breast cancer, the aromatase activity has been measured in tumour and fatty tissues dissected at specific sites from the breasts of 16 patients. Activity was measured after extensive purification of the product formed. Results, expressed in fmol/g of tissue, did not show a higher activity in the affected vs the non-affected quadrants. In the tumours, higher activities were found when expressed per g of tissue. Per mg of DNA, an indicator of the number of cells, tumour enzymatic activity was lower than in fatty tissues. The relations between the products of aromatase, oestrone and oestradiol in the various tissues point to the importance of additional enzymatic processes, especially of the reductive 17 beta-oestradiol dehydrogenase, in the accumulation of high quantities of oestradiol in the malignant tissue.

Multiple functions of aromatase and the active site structure; aromatase is the placental estrogen 2-hydroxylase.

Androgen aromatase was found to also be estrogen 2-hydroxylase. The substrate specificity among androgens and estrogens and multiplicity of aromatase reactions were further studied. Through purification of human placental microsomal cytochrome P-450 by monoclonal antibody-based immunoaffinity chromatography and gradient elution on hydroxyapatite, aromatase and estradiol 2-hydroxylase activities were co-purified into a single band cytochrome P-450 with approx. 600-fold increase of both specific activities, while other cytochrome P-450 enzyme activities found in the microsomes were completely eliminated. The purified P-450 showed M(r) of 55 kDa, specific heme content of 12.9 +/- 2.6 nmol.mg-1 (+/- SD, n = 4), reconstituted aromatase activity of 111 +/- 19 nmol.min-1.mg-1 and estradiol 2-hydroxylase activity of 5.85 +/- 1.23 nmol.min-1.mg-1. We found no evidence for the existence of catechol estrogen synthetase without concomitant aromatase activity. The identity of the P-450 for the two different hormone synthetases was further confirmed by analysis of the two activities in the stable expression system in Chinese hamster ovarian cells transfected with human placental aromatase cDNA, pH beta-Aro. Kinetic analysis of estradiol 2-hydroxylation by the purified and reconstituted aromatase P-450 in 0.1 M phosphate buffer (pH 7.6) showed Km of 1.58 microM and Vmax of 8.9 nmol.min-1.mg-1. A significant shift of the optimum pH and Vmax, but not the Km, for placental estrogen 2-hydroxylase was observed between microsomal and purified preparations. Testosterone and androstenedione competitively inhibited estradiol 2-hydroxylation, and estrone and estradiol competitively inhibited aromatization of both testosterone and androstenedione. Estrone and estradiol showed Ki of 4.8 and 7.3 microM, respectively, for testosterone aromatization, and 5.0 and 8.1 microM, respectively, for androstenedione aromatization. Androstenedione and testosterone showed Ki of 0.32 and 0.61 microM, respectively, for estradiol 2-hydroxylation. Our studies showed that aromatase P-450 functions as estrogen 2-hydroxylase as well as androgen 19-, 1 beta-, and 2 beta-hydroxylase and aromatase. The results indicate that placental aromatase is responsible for the highly elevated levels of the catechol estrogen and 19-hydroxyandrogen during pregnancy. These results also indicate that the active site structure holds the steroid substrates to face their beta-side of the A-ring to the heme, tilted in such a way as to make the 2-position of estrogens and 19-, 1-, and 2-positions of androgens available for monooxygenation.

Cloning of two mRNA variants of brain aromatase cytochrome P450 in rainbow trout (Oncorhynchus mykiss Walbaum).

This work describes the molecular cloning of the cDNA encoding the rainbow trout (Oncorhynchus mykiss Walbaum) brain cytochrome P450arom by means of reverse transcriptase and polymerase chain reaction (RT-PCR) and 5'- and 3'-rapid amplification of cDNA ends (RACE) analyses. The results obtained demonstrate that, as in other teleost fishes, the trout genome contains, besides the gene previously identified in the ovary, a second CYP19 gene (CYP19B) expressed at high level in the brain. Moreover, two P450aromB mRNAs, forms I and II, were found to be transcribed in trout. Form I (1816 sequenced nt) contains an open reading frame (ORF) of 1464b, a 5'-untranslated terminal region (UTR) of 124b and at least 228b in the 3'-UTR (incomplete, as the polyadenylation signal was not determined). Form II (1930 sequenced nt) contains an ORF of 1362b, a 5'-UTR of 340b and the same 3'-UTR as form I. Form II lacks the first 34 amino acids of form I, corresponding to the membrane-anchoring segment, whereas the sequence of the remaining coding region is almost the same in the two forms, resulting in proteins of 454 and 488 amino acids, respectively. Whether the two transcripts derive from the same gene by alternative splicing or are encoded by different CYP19B genes remains to be clarified. On Northern blot analyses with brain and ovary specific ORF probes and poly(A)(+)-enriched RNAs from trout ovary and brain, a transcript of about 2.6kb was identified in the ovary, as expected, whereas the full-length mRNA of brain P450arom is about 3.8kb. The brain form is expressed in the brain and gonads, whereas expression in peripheral tissues is limited mostly to the gills. The two trout CYP19 genes are not equivalent in tissue-specific expression, indicating the possibility of distinct promoters and regulatory mechanisms.

Aromatase cytochrome P-450. Purification and characterization of the enzyme from human placenta.

Aromatase cytochrome P-450 (P-450arom) was purified from human placental microsomes. Preparations exhibit a single major band of approximately 55 kDa on SDS-polyacrylamide gel electrophoresis and have a specific content of 11-13 nmol P-450/mg protein. The purified enzyme exhibits spectral properties typical of ferric and ferrous forms of cytochromes P-450. Full enzymatic activity can be reconstituted with rabbit liver P-450 reductase, and catalytic characteristics similar to aromatase in microsomes are observed. Rabbit antibodies to purified P-450arom were affinity purified and show high specificity and sensitivity on immunoblots.

Properties of the aromatase system associated with the mitochondrial fraction of human placenta.

The aromatase system associated with the mitochondrial fraction of human term placenta, present at 35--50% the specific activity of the microsomal enzyme, is substantially the same as the microsomal enzyme as determined by the following: 1) The rate of aromatization of androstenedione, 19-nortestosterone, and 16alpha-hydroxytestosterone in mitochondria was a nearly constant proportion of the microsomal rate; 2) Sensitivity to carbon monoxide was the same; 3) The magnitude of cytochrome P-450 Type I spectral interactions with androgen substrates was a constant proportion in mitochondria and microsomes; 4) Sensitivity to an antibody raised against hepatic microsomal NADPH-cytochrome c reductase was the same. When inner and outer mitochondrial membrane subfractions were prepared, the predominant aromatase activity was associated with the outer membrane preparation. This aromatase activity could not be accounted for by microsomal contamination as determined by inosine diphosphatase activity, a microsomal marker. After correction, the rate of aromatization in the outer membrane preparation was almost six times that in the inner membranes and three times that of the whole mitochondrial fraction.