Francis L. Bellino

Estrogen synthetase (aromatase). The cytochrome P-450 component of the human placental enzyme is a glycoprotein

Estrogen synthetase (aromatase) is a cytochrome P-450 enzyme system which converts androgens to estrogens. Although this enzyme has been purified and the cDNA-derived amino acid sequence elucidated, very little is known regarding post-translational modifications of this physiologically crucial enzyme. We show here that the cytochrome P-450 component, P-450ES, purified from human term placental microsomes, is a glycoprotein based on the following evidence: its molecular weight is decreased following treatment with endoglycosidase F, concanavalin A-biotin specifically binds to this protein immobilized on nitrocellulose, and its oligosaccharide composition is consistent with a single N-linked fucosylated complex type carbohydrate chain. In a reconstitution system, the aromatase activity using the endoglycosidase F-treated P-450ES was reduced by about 35-40% relative to the native form, regardless of whether androstenedione or testosterone was used as substrate.

Solubilization of estrogen synthetase from human term placental microsomes using detergents.

Abstract The microsomal fraction of term placenta was incubated with a range of concentrations of several bile acids and non-ionic detergents (deoxycholate, cholate, taurocholate, Triton X-100, Tween 80, Lubrol PX, Lubrol WX and digitonin) and then centrifuged at 120,000 g for 1 h. Aromatase activity was progressively depleted from the pellet with increasing detergent concentration for each detergent except Tween 80 (up to 5% concentration). Since a cytochrome P-450 monoxygenase system is assumed to be involved in androgen aromatization, two detergents, deoxycholate and Triton X-100, were also examined for their ability to remove NADPH cytochrome c reductase activity and cytochrome P-450 from the microsomes. Although the cytochrome was removed from microsomes at detergent concentrations similar to those for aromatase, approximately 2- to 3-fold higher detergent concentrations were required to deplete the microsomes of reductase activity. Each detergent used inhibited aromatase activity in the microsomal homogenate in a concentration-dependent manner that roughly paralleled the concentration-dependent depletion of aromatase activity from the pellet. Aromatase activity was measured in the supernatant (120,000 g-h) from the cholate- and deoxycholate-treated microsomes by diluting the supernatant to lower detergent concentrations. Significant amounts of aromatase activity could not be measured in the 120,000 g-h supernatant from non-ionic detergent-treated microsomes even after dilution to lower detergent concentrations. Dilution of the cholate- and deoxycholate-soluble prepaations to lower detergent concentrations resulted in an aromatase-active precipitate with a 3-fold higher specific activity than the original microsomes. Deoxycholate- and cholate-solubilized aromatase activity is included in a Sepharose 6B column, eluting at an approximate molecular weight of 100,000 to 120,000 daltons.

Placental estrogen synthetase (aromatase): evidence for phosphatase-dependent inactivation.

The acute regulation of estrogen synthetase (aromatase), the cytochrome P450 enzyme system responsible for estrogen production, is not well explored. We report here that aromatase, but not NADPH-cytochrome c (P450) reductase, activity from human term placental microsomes decreased when incubated in phosphate-free buffer at 37 degrees C. Aromatase activity was stabilized by phosphate buffer or by the phosphatase inhibitors tartaric acid or EDTA, but not NaF, in phosphate-free buffer. Alkaline phosphatase also inhibited aromatase in phosphate-free buffer relative to phosphate buffer, but the inactivation appears to be due primarily to proteolytic solubilization of NADPH-cytochrome c reductase from the microsomes by proteases within the alkaline phosphatase preparation. Based on these data, we suggest that the cytochrome P450 component of aromatase may be regulated acutely by phosphorylation-dependent processes.

Antisera against estrogen synthetase from human placental microsomes. Antibody characterization and cross-reactivity studies in other organs.

Antibodies were obtained against both protein components of the cytochrome P-450 enzyme system responsible for estrogen biosynthesis, estrogen synthetase (aromatase), from human placental microsomes. The antiserum against the NADPH-cytochrome P-450 reductase component (antiserum denoted RE-DFBIV) gave a single major band at the Mr of the authentic enzyme by immunoblotting after electrophoretic separation of SDS-solubilized microsomes and inhibited both the reductase and aromatase activities in human placental and endometrial microsomes (Tseng, L. and Bellino, F.L. (1985) J. Steroid Biochem. 22, 555-557) and in homogenates of cultured aromatase-stimulated human endometrial stromal cells and human ovarian microsomes. The antiserum against the cytochrome P-450 component of aromatase (antiserum denoted P45FBIII) also gave a single band at the Mr of the authentic protein by immunoblotting after electrophoresis, and inhibited aromatase activity in homogenates of human placental microsomes, ovarian and decidual particulate fractions and cultured aromatase-stimulated endometrial stromal cells. This antiserum had no effect on NADPH-cytochrome c reductase activity in any of the systems studied. We conclude that these antiserum preparations separately recognize the NADPH-cytochrome P-450 reductase and cytochrome P-450 components of aromatase in human placenta, ovary, decidua and endometrium. Epitopes on these aromatase component proteins involved in enzyme activity are shared among these various human tissue sources.

Immunologic identification of the aromatase enzyme system in human endometrium

Confluent human endometrial stromal cells were cultured in medium with no hormone or supplemented with medroxyprogesterone acetate (MPA), estradiol (E2), and porcine relaxin (RLX) for 5 days. These stromal cells were then labeled with [35S]methionine for 3 h. The radioactive proteins in the particulate fraction of cell homogenate were extracted by detergent and incubated with antisera to purified placental aromatase cytochrome P-450 (P-450arom) and NADPH-cytochrome P-450 reductase to isolate the radio-labeled aromatase enzyme components. Analysis of the radio-labeled protein, isolated by antibody to the cytochrome P-450arom from different preparations (P45FBIII or R-8-2) showed a major band at molecular weight 54k on SDS polyacrylamide gel electrophoresis (SDS-PAGE). The intensity of 54k band was stronger in hormone treated stromal cells than that of control in parallel with the increase of aromatase activity. The radio-labeled protein isolated by anti-NADPH cytochrome P-450 reductase, REDFBIV, showed a major band at the molecular weight 73k on SDS-PAGE with comparable intensity in control and hormone treated samples. Thus, the apparent molecular weights of endometrial cytochrome P-450arom and cytochrome P-450 reductase were identical to placental aromatase enzyme system. When a secretory endometrium and a decidua were labeled with [35S]methionine, the cytochrome P-450arom was detected only in the decidua. NADPH cytochrome P-450 reductase was detected both in the endometrium and the decidua. These results show that antisera to placental aromatase enzyme system cross reacts with the endometrial aromatase enzyme components. The synthesis of cytochrome P-450arom was stimulated by MPA, E2 and RLX while the synthesis of the NADPH-cytochrome P-450 reductase aromatase component was not affected by the hormone.

Estrogen synthetase stimulation by hemin in human choriocarcinoma cell culture

The ability of hemin to stimulate estrogen synthetase (aromatase) in cultured human trophoblast cells and in cellular homogenates was investigated and compared with aromatase stimulation by dibutyryl cAMP [(Bu)2 cAMP]. Cells grown with hemin for 24 h, or homogenates incubated for 45 min with hemin, showed maximal aromatase stimulation (150 to 200% of activities in the absence of hemin) at 25 microM and 0.1 microM, respectively. Aromatase stimulation in culture by 25 microM hemin was observed within 4 h after hemin addition, while (Bu)2 cAMP required more than 6 h. Intracellular heme and porphyrin levels were higher (160 to 185%) in 96 h (Bu)2 cAMP-grown cells than control cells.

Oestrogen synthetase (Aromatase) and hormone secretion in primary cultures of human placental trophoblast cells. Effects of cyclic AMP addition at the start of culture in attached and unattached cell populations

Term placental trophoblast cells, released by trypsin digestion of placental villi, purified on a Percoll gradient and grown in serum-containing medium, differentiate within 24 to 48 h in culture from mononucleated cytotrophoblast-like cells at the start of culture to highly multinucleated giant (syncytiotrophoblast-like) cells that are more active in hormonogenesis. To determine the changes in hormone biosynthesis and secretion that occur early in the trophoblast differentiation process in vitro, freshly isolated placental cells were cultured in Dulbeccos Modified Eagles Medium (DMEM) containing 20 per cent FBS, in the presence and absence of cAMP for up to 48 h. Cell attachment and growth, oestrogen synthetase (aromatase) activity in attached and unattached cells, and secretion of human chorionic gonadotropin (hCG) and progesterone were studied. The aromatase specific activity, low in freshly isolated cells, increased fourfold in attached cells by 3 h, and achieved a 10- to 15-fold increase by 40 to 48 h. In attached cells grown with cAMP, aromatase activity was further stimulated by about fourfold, relative to the control. The aromatase activity of the unattached cells removed from the culture dishes at various times up to 48 h showed a biphasic response: the activity decreased by 18 h and then increased back to the fresh cell levels. The effect of cAMP on aromatase in these unattached cells was manifested by a two-fold stimulation of activity by 18 h, relative to control unattached cells. Secretion of hCG from both attached and unattached cells remained at a low level (less than 200 ng/mg protein) in control cells; in the presence of cAMP, hCG secretion was stimulated by tenfold after 40 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of aromatase and NADPH cytochrome C reductase activities in human endometrium by the human placental NADPH cytochrome C reductase antiserum

The cross-reactivity of human placental microsomal NADPH-cytochrome c reductase antiserum, REDFBIV, against the endometrial reductase alone and as a component of the endometrial aromatase was investigated. Human endometrial particulate fractions were incubated with various amounts of REDFBIV for 1 h at 4 degrees C and both enzyme activities were measured at the end of incubation. The extent of inhibition of these endometrial enzymes was compared with the ability of this antiserum to inhibit the placental microsomal reductase and aromatase activities. The antiserum effectively inhibited the activities of both enzymes in both tissues in a dose dependent manner with aromatase activity inhibited to a greater extent than reductase activity. These results indicate the antiserum to the placental microsomal NADPH-cytochrome c reductase component of aromatase recognizes the reductase component of the aromatase enzyme system in endometrium.

Human placental estrogen synthetase (aromatase). Effect of environment on the kinetics of protein-protein and substrate-protein interactions and the production of 19-oxygenated androgen intermediates in the purified reconstituted cytochrome P450 enzyme system

Estrogen synthetase (aromatase) catalyzes the conversion of androgen into estrogen via two hydroxylations at C19 and a subsequent C19-10 lyase reaction. We report here the results of a reconstitution study using a highly purified aromatase cytochrome P450 monooxygenase enzyme system, with both protein components (cytochrome P450 and NADPH-cytochrome P450 reductase) obtained from human term placental microsomes. By varying one of the components (amounts of cytochrome P450, NADPH-cytochrome P450 reductase, or androgen substrate) as the other two were held constant in four different environments (phospholipid, non-ionic detergent, mixture of phospholipid and non-ionic detergent and buffer alone), we obtained evidence supporting the following conclusions. The reconstituted enzyme is more active and the protein components exhibit much lower apparent Km values in the detergent and/or lipid environment compared with buffer alone. Although the apparent Km and Vmax values for each aromatase protein component differ significantly in most cases with the particular limiting component and environment, the catalytic efficiency (Kcat/Km) was independent of the limiting protein component and varied with the environment only (highest in the lipid-detergent mixture and lowest in lipid alone). When the concentration of androgen substrate (androstenedione or testosterone) was varied at constant amounts of the aromatase protein components (NADPH-cytochrome P450 reductase saturating), the Km was lower and the Vmax was higher for adrostenedione. The specificity constant (Vmax/Km) was a function of the reconstitution environment (highest in lipid alone and lowest in detergent alone) and was, on average, about 4-fold higher for androstenedione in a particular environment. The extent of production of 19-oxygenated androgen intermediates (19-hydroxy and 19-oxo androstenedione) was examined at three different levels of aromatase cytochrome P450 (subsaturating, saturating, super-saturating) relative to the NADPH-cytochrome P450 reductase component in the three different hydrophobic environments using androstenedione as substrate. Both 19-oxygenated androgens, each made in comparable amounts relative to control, were isolatable in greatest amounts under cytochrome P450 super-saturating conditions in the detergent-lipid mixed environment, and in least amounts under cytochrome P450 subsaturating conditions in the lipid-only environment. Based on these data, we propose that 19-oxygenated androgen intermediates are biosynthesized sequentially in a step-wise fashion as the cytochrome P450 and NADPH-cytochrome P450 reductase form transient complexes, and that the amount of isolatable 19-oxygenated androgen is proportional to the amount of excess cytochrome P450 component.

Estrogen synthetase (aromatase). Affinity purification of antibody against the cytochrome P450 component

To procure an affinity gel capable of purifying antibody against the cytochrome P450 component of estrogen synthetase (P450ES), we coupled purified P450ES to agarose supports. OUr purpose was to compare two differently-activated agarose gels (Affi-Gel 15 and Tresyl-activated Sepharose) with the same P450ES preparation to compare the efficiency of coupling and the yield of purified antibody. Using supplier-directed protocols to covalently attach P450ES to each of the supports, and identical procedures to bind and elute anti-P450ES, we reached the following conclusions. Tresyl-activated Sepharose bound greater amounts of antigen than Affi-Gel 15 based on the amount of residual antigen after the coupling procedure and the amount of bound antigen detected by an ELISA-type method. However, both ligand-coupled supports yielded comparable amounts of purified anti-P450ES at a 48-fold purification relative to the starting IgG preparation.