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·mmg⁻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 β-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 K_m of 1.58 μM and V_max of 8.9 nmol·min⁻¹·mg⁻¹. A significant shift of the optimum pH and V_max, but not the K_m, 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 K_i of 4.8 and 7.3 μM, respectively, for testosterone aromatization, and 5.0 and 8.1 μM, respectively, for androstenedione aromatization. Androstenedione and testosterone showed K_i of 0.32 and 0.61 μM, respectively, for estradiol 2-hydroxylation. Our studies showed that aromatase P-450 functions as estrogen 2-hydroxylase as well as androgen 19-, 1β-,and 2β-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 ssubstrates to face their β-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.     

Competitive product inhibition of aromatase by natural estrogens

In order to better understand the function of aromatase, we carried out kinetic analyses to asses the ability of natural estrogens, estrone (E₁), estradiol (E₂), 16-OHE₁, and estriol (E₃), to inhibit aromatization. Human placental microsomes (50 μg protein) were incubated for 5 min at 37°C with [1β-³H]testosterone (1.24 × 10³ dpm ³H/ng, 35–150 nM) or [1β-³H,4-¹⁴C]androstenedione (3.05 × 10³ dpm ³H/ng, ³H/¹⁴C = 19.3, 7–65 nM) as substrate in the presence of NADPH, with and without natural estrogens as putative inhibitors. Aromatase activity was assessed by tritium released to water from the 1β-position of the substrates. Natural estrogens showed competitive product inhibition against androgen aromatization. The K_i of E₁, E₂, 16-OHE₁, and E₃ for testosterone aromatization was 1.5, 2.2, 95, and 162 μM, respectively, where the K_m of aromatase was 61.8 ± 2.0 nM (n = 5) for testosterone. The K_i of E₁, E₂, 16-OHE₁, and E₃ for androstenedione aromatization was 10.6, 5.5, 252, and 1182 μM, respectively, where the K_m of aromatase was 35.4 ± 4.1 nM (n = 4) for androstenedione. These results show that estrogens inhibit the process of andrigen aromatization and indicate that natural estrogens regulate their own synthesis by the product inhibition mechanism in vivo. Since natural estrogens bind to the active site of human placental aromatase P-450 complex as competitive inhibitors, natural estrogens might be further metabolized by aromatase. This suggests that human placental estrogen 2-hydroxylase activity is catalyzed by the active site of aromatase cytochrome P-450 and also agrees with the fact that the level of catecholestrogens in maternal plasma increases during pregnancy. The relative affinities and concentration of androgens and estrogens would control estrogen and catecholestrogen biosynthesis by aromatase.     

Biochemical and biophysical studies on cytochrome aa3 VIII. Effect of cyanide on the catalytic activity

1. 1. Cyanide inhibits the catalytic activity of cytochrome aa₃ in both polarographic and spectrophotometric assay systems with an apparent velocity constant of 4·10³ M⁻¹·s⁻¹ and a K_i that varies from 0.1 to 1.0 μM at 22 °C, pH 7·3.

2. 2. When cyanide is added to the ascorbate-cytochrome c-cytochromeaa₃−O₂ system a biphasic reduction of cytochrome c occurs corresponding to an initial K_i of 0.8 μM and a final K_i of about 0.1 μM for the cytochrome aa₃−cyanide reaction.

3. 3. The inhibited species (a²+a₃³⁺HCN) is formed when a²⁺a₃³⁺ reacts with HCN, when a²⁺a₃²⁺HCN reacts with oxygen, or when a³⁺a₃³⁺HCN (cyano-cytochrome aa₃) is reduced. Cyanide dissociates from a²⁺a₃³⁺HCN at a rate of 2·10⁻³ s⁻¹ at 22 °C, pH 7.3.

4. 4. The results are interpreted in terms of a scheme in which one mole of cyanide binds more tightly and more rapidly to a²⁺a₃³⁺ than to a³⁺a₃³⁺.

Interaction of sialosyl cholesterol with the cell surface of rat astrocytes and its biological activities

The interaction between sialosyl cholesterol (- or neuraminyl cholesterol, - or β-SC) and the plasma membrane of astrocytes was investigated by the use of ¹⁴C-labeled - or β-SC. Both - and β-SC were dose-dependently and time-dependently bound to rat astrocytes. The Scatchard plot analyses showed that rat astrocytes bound apparently 9.69 × 10⁹ molecules of both -SC/cell (apparent K_d = 2.29 × 10⁻⁵ M) and β-SC/cell (apparent K_d = 5.39 × 10⁻⁵ M) at 37°C. Both the binding of -SC to astrocytes and the subsequent inhibition of DNA synthesis were decreased at the low temperature (4°C), and also suppressed by serum proteins including albumin. One molecule of bovine serum albumin (BSA) bound 2.3 molecules of -SC with the slightly lower K_d-value (8.03 × 10⁻⁶ M) than that for the binding site on astrocytes. BSA not only suppressed the -SC-binding to astrocytes but also increased its release from the cells to the culture media. Gangliosides such as GM1 and GM3 unaffected the -SC-binding, promoted the small release of -SC from the cell surface, and inhibited the morphological changes of astrocytes induced by -SC. The mechanism of -SC-binding to cultured astrocytes with reference to the effects of serum or gangliosides is discussed.     

Effect of nomegestrol acetate on estrone-sulfatase and 17β-hydroxysteroid dehydrogenase activities in human breast cancer cells

It is well recognized that estradiol (E₂) is one of the most important hormones supporting the growth and evolution of breast cancer. Consequently, to block this hormone before it enters the cancer cell or in the cell itself, has been one of the main targets in recent years. In the present study we explored the effect of the progestin, nomegestrol acetate, on the estrone sulfatase and 17β-hydroxy-steroid dehydrogenase (17β-HSD) activities of MCF-7 and T-47D human breast cancer cells. Using physiological doses of estrone sulfate (E₁S: 5 × 10⁻⁹ M), nomegestrol acetate blocked very significantly the conversion of E₁S to E₂. In the MCF-7 cells, using concentrations of 5 × 10⁻⁶ M and 5 × 10⁻⁵ M of nomegestrol acetate, the decrease of E₁S to E₂ was, respectively, −43% and −77%. The values were, respectively, −60% and −71% for the T-47D cells. Using E₁S at 2 × 10⁻⁶ M and nomegestrol acetate at 10⁻⁵ M, a direct inhibitory effect on the enzyme of −36% and −18% was obtained with the cell homogenate of the MCF-7 and T-47D cells, respectively. In another series of studies, it was observed that after 24 h incubation of a physiological concentration of estrone (E₁: 5 × 10⁻⁹ M) this estrogen is converted in a great proportion to E₂. Nomegestrol acetate inhibits this transformation by −35% and −85% at 5 × 10⁻⁷ M and 5 × 10⁻⁵ M, respectively in T-47D cells; whereas in the MCF-7 cells the inhibitory effect is only significant, −48%, at 5 × 10⁻⁵ M concentration of nomegestrol acetate. It is concluded that nomegestrol acetate in the hormone-dependent MCF-7 and T-47D breast cancer cells significantly inhibits the estrone sulfatase and 17β-HSD activities which converts E₁S to the biologically active estrogen estradiol. This inhibition provoked by this progestin on the enzymes involved in the biosynthesis of E₂ can open new clinical possibilities in breast cancer therapy.     

New herbicide-binding site in the photosynthetic electron-transport chain: Competitive herbicide binding at the photosystem I phylloquinone-(vitamin K1)-binding site

The binding of herbicides to the phylloquinone-(primary electron acceptor A₁)-binding site in green plant photosystem (PS) I reaction centers is shown. Dissociation constants (K_d) of various herbicides to the phylloquinone-binding site were estimated by analyzing their competitive inhibition of the reconstitution of the phylloquinone analogue, menadione (vitamin K₃), to the phylloquinone-extracted spinach PS I particles. The phylloquinone-binding site was found to bind o-phenanthroline (K_d = 1.2 × 10⁻⁴ M), but only weak binding was observed with atrazine (K_d > 10⁻² M), although both are known to bind specifically to the quinone-(Q_B)-binding site in reaction centers of purple photosynthetic bacteria or PS II. The inhibitors of the cytochrome b/c₁(ƒ) complex, myxothiazol (K_d=9.5 × 10⁻⁶ M) or antimycin A (K_d = 2.8 × 10⁻⁶ M), also strongly bound to the phylloquinone site. This is the first report showing that the PS I reaction center complex also has a herbicide-binding site, although the site is probably not sensitive in vivo to these herbicides due to its higher affinity for phylloquinone than herbicides. The inhibitor specificity of the PS I phylloquinone site is different from that of the other quinone-functioning sites in the photosynthetic or respiratory electron-transfer chain, suggesting it to have a unique structure.     

Regulation of sex-specific formation of oestrogen in brain development: Endogenous inhibitors of aromatase

Brain sexual differentiation occurs during steroid-sensitive phases in early development, and is affected particularly by exposure to oestrogens formed in the brain by aromatisation of androgen. The organisational effects of oestrogen result in male-specific neuronal morphology, control of reproductive behaviour, and patterns of gonadotrophin secretion. A question which still has to be resolved is what determines changes in aromatase activity effective for the differentiation of sexually dimorphic brain development during sensitive periods of growth. In the mouse, a sex difference exists at early stages of embryonic development in aromatase-containing neurones of the hypothalamus. The embryonic aromatase system is regulated later in foetal development by androgens. Testosterone treatment increases the numbers of aromatase-immunoreactive hypothalamic neuronal cell bodies. Kinetic evidence from studies on the avian brain suggest that endogenous steroid inhibitors of aromatase, probably formed within neuroglia, also have a role in the control of oestrogen production. Inhibitory kinetic constant determination of endogenous androgenic metabolites formed in the brain showed that preoptic aromatase is potently inhibited by 5-androstanedione (K_i = 6 nM) and less strongly by 5β-dihydrotestosterone (K_i = 350 nM). Regulation by steroidal and possibly non-steroidal inhibitors may contribute to the special characteristics and plasticity in aromatase activity which develops at certain stages in ontogeny.     

Uptake of corticosterone into isolated rat liver cells: Possible involvement of Na/K-ATPase

Isolated rat hepatocytes posses a saturable glucocorticoid uptake system with high affinity (K_d value = 2.8 ± 0.7 × 10⁻⁸ M; 318,000 ± 80,000 binding sites per cell; 317 fmol/mg protein). The initial rates of uptake decrease by about 30–40% if the cells are incubated simultaneously with [³H]corticosterone and either SH-reagents (N-ethylmaleimide and p-chloromercuriphenylsulphonate, 1 mM), metabolic inhibitors (2,4-dinitrophenol, 1 mM; and antimycin, 0.1 mM) or the Na⁺/K⁺-ATPase-inhibitors, ouabain and quercetine. These Na⁺/K⁺-ATPase-blockers exert half-maximal inhibition at 3 × 10⁻⁷ and 3 × 10⁻⁶ M, respectively. A slight increase in K⁺ concentration and a corresponding decrease in Na⁺ in the medium leads to a significant reduction in the initial uptake rate. The uptake system from the rat hepatocytes shows a clear steroid specificity, being different from the intracellular receptor. Corticosterone and progesterone are the strongest competitors, cortisol, 5- and 5β-dihydrocorticosterone, 11-deoxycorticosterone, cortisone and testosterone have an intermediate effect and only weak competition is exerted by dexamethasone and by the mineralocorticoid, aldosterone. Estradiol and estrone sulphate as well as the synthetic glucocorticoid triamcinolone acetonide are unable to inhibit initial corticosterone uptake.     

Fuscin, an inhibitor of mitochondrial SH-dependent transport-linked functions

1. 1. Fuscin, a mould metabolite, is a colored quinonoid compound which reacts readily with −SH groups to give colorless addition derivatives.

2. 2. Binding of fuscin to mitochondria has been monitored spectrophotometrically. Fuscin binding is prevented by −SH reagents such as N-ehylmaleimide, N-Methylmaleimide, mersalyl or p-chloromercuribenzoate. Conversely, fuscin prevents the binding of −SH reagents as shown with N-[¹⁴C]ethylmaleimide. Once bound to mitochondria, fuscin is not removable by washing of mitochondria.

3. 3. High affinity-fuscin binding sites (K_d = 1 μM, N = 4–8 nmoles/mg protein) are present in whole mitochondria obtained from rat heart, rat liver, pigeon heart or yeast (Candida utilis). They are lost upon sonication but are still present in digitonin inner membrane + matrix vesicles. On the other hand, lysis of mitochondria by Triton X-100 does not increase the number of high affinity binding sites indicating that all these sites are accessible to fuscin in whole mitochondria. The number of fuscin high affinity sites appears to correlate with the glutathione content of mitochondrial preparations.

4. 4. Fuscin as well as N-ethylmaleimide and avenaciolide are penetrant SH-reagents;

5. 5. Fuscin interferes with the ADP-stimulated respiration of mitochondria on NAD-linked substrates, several functions of the mitochondrial respiratory apparatus being inhibited by fuscin in a non-competitive manner, but to various extents: (a) The electron transfer chain (K_i in the range of 0.1 mM); (b) the lipoamide dehydrogenase system (K_i = 5–10 μM); (c) the transport systems of phosphate (K_i ≈ 20 μM) and of glutamate (K_i = 3–5 μM); (d) the ADP transport, indirectly (K_i ≈ 10 μM).

6. 6. Like N-ethylmaleimide, fuscin inhibits the glutamate-OH⁻ carrier, the inhibition of that carrier bringing about an apparent increase of aspartate entry in glutamate-loaded mitochondria by the glutamate-aspartate carrier.

7. 7. The inhibition of phosphate transport by fuscin probably accounts for the inhibition of the reduction of endogenous NAD by succinate in intact pigeon heart mitochondria.

8. 8. By binding the −SH groups of mitochondrial membrane specifically unmasked by addition of micromolar amounts of ADP, fuscin, like N-ethylmaleimide, prevents the functioning of ADP translocation.

9. 9. Because of their specific and analogous effects on some well defined mitochondrial functions such as glutamate transport and ADP transport, fuscin and N-ethylmaleimide can be distinguished from other −SH reagents. The lipophilic nature of fuscin and N-ethylmaleimide which accounts for the accessbility of these compounds to hydrophobic sites in the mitochondrial membrane or on the matrix side of this membrane may be partly responsible for their characteristic inhibitory effects on mitochondrial functions.

Nitric oxide stimulates prostaglandin synthesis in cultured rabbit gastric cells

Both prostaglandins (PGs) and nitric oxide (NO) have cytoprotective and hyperemic effects in the stomach. However, the effect of NO on PG synthesis in gastric mucosal cells is unclear. We examined whether sodium nitroprusside (SNP), a releaser of NO, stimulates PG synthesis in cultured rabbit gastric mucus-producing cells. These cells did not release NO themselves. Co-incubation with SNP (2 × 10⁻⁴, 5 × 10⁻⁴, 10⁻³ M) increased PGE₂ synthesis, and SNP (10⁻³ M) increased PGI₂ synthesis in these cells. Hemoglobin, a scavenger of NO, (10⁻⁵ M) eliminated the increase in PGE₂ synthesis by SNP, but methylene blue, an inhibitor of soluble guanylate cyclase, (5 × 10⁻⁵ M) did not affect the increase in PGE₂ synthesis by SNP. 8-bromo guanosine 3′ : 5′-cyclic monophosphate (8-bromo cGMP), a cGMP analogue, (10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³ M) did not affect PGE₂ synthesis. These findings suggest that NO increased PGE₂ and PGI₂ synthesis via a cGMP-independent pathway in cultured rabbit gastric cells.     

Two distinct types of cell surface folic acid-binding proteins in Dictyostelium discoideum

Folic acid is degraded too fast by Dictyostelium discoideum to study binding of this ligand to cell surface binding proteins. Folate deaminase activity was inhibited in the presence of 3.3 × 10⁻⁴ M 8-azaguanine. This inhibitor enabled us to detect two folate binding proteins. One type bound folic acid and deamino-folic acid with the same affinity (K_0.5 = 3–6 × 10⁻⁷ M) and apparently negative cooperativity. Binding to only this type was observed if 8-azaguanine was omitted. The second type bound folic acid noncooperatively with K_d = 7 × 10⁻⁷ M. Deamino-folic acid did not compete even at a 1000-fold excess. This type may correspond to the chemotactic receptor.     

Inhibition of steroid sulphatase activity by steroidal methylthiophosphonates: Potential therapeutic agents in breast cancer

The hydrolysis of steroid sulphates, by steroid sulphatase, is an important source of oestrogenic steroids (oestrone, oestradiol and 5-androstene-3β,17β-diol) which are found in tumours. In the present study, we have examined the effect of dehydroepiandrosterone-3-O-methylthiophosphonate (DHA-3-MTP), pregnenolone-3-O-methylthiophosphonate (pregnenolone-3-MTP) and cholesterol-3-O-methylthiophosphonate (cholesterol-3-MTP) on the inhibition of oestrone sulphatase as well as DHA sulphatase activities in intact MCF-7 breast cancer cells and in placental microsomes. All three methylthiophosphonates significantly (P< 0.01) inhibited the hydrolysis of oestrone sulphate (E₁ S) in intact MCF-7 cells (31–85% inhibition at 1 μM and 53–97% inhibition at 10 μM). Significant inhibition of DHA sulphatase was also achieved. At a concentration of 50 μM, all three compounds inhibited the hydrolysis of dehydroepiandrosterone sulphate (DHAS) by > 95%. Using human placental microsomes, the K_m and V_max of E₁S were determined to be 8.1 μM and 43 nmol/h/mg protein. The corresponding K_i values for DHA-3-MTP, pregnenolone-3-MTP and cholesterol-3-MTP were found to be 4.5, 1.4 and 6.2 μM, respectively. Such inhibitors which are resistant to metabolism may have considerable potential as therapeutic agents and may have additional advantage over aromatase inhibitors in also reducing tumour concentrations of the oestrogenic steroid, 5-androstene-3β,17β-diol, by inhibiting the hydrolysis of DHAS.     

Differential inhibition of dehydrogenase and 5-ene→4-ene isomerase activities of purified 3β-hydroxysteroid dehydrogenase. Evidence for two distinct sites

The success in synthesis of [³H]5-androstene-3,17-dione, the intermediate product in the transformation of DHEA to 4-androstenedione by 3β-hydroxysteroid dehydrogenase/ 5-ene→4-ene isomerase (3β-HSD) offers the opportunity to determine whether or not the two activities reside in one active site or in two closely related active sites. The finding that N,N-dimethyl-4-methyl-3-oxo-4-aza-5-androstane-17β-carboxamide (4-MA) inhibits competitively and specifically the dehydrogenase activity whereas a non-competitive inhibition type with a K_i value 1000 fold higher was observed for the isomerase activity, indicated that dehydrogenase and isomerase activities belong to separate sites. Using 5-dihydro-testosterone and 5-androstane-3β,17β-diol, exclusive substrates for dehydrogenase activity, it was shown that dehydrogenase is reversible and strongly inhibited by 4-MA and that thus the irreversible step in the transformation of DHEA to 4-androstenedione is due to the isomerase activity.     

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 (K_i values) were 10 times greater than the 2,19-methylene-bridged androstenedione constant (K_i = 35–70 nM). However, expansion of the 2,19-carbon bridge to ethylene increased aromatase affinity by 10-fold (K_i = 2 nM). Substitution pf a methylene group with oxygen and sulfur in this expanded bridge resulted in K_i 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 τ₅₀ values, the t_1/2 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 K_i values.     

Cortisol metabolism in vitro—III. Inhibition of microsomal 6β—hydroxylase and cytosolic 4-ene-reductase

The in vitro metabolism of cortisol in human liver fractions is highly complex and variable. Cytosolic metabolism proceeds predominantly via A-ring reduction (to give 3,5β-tetrahydrocortisol; 3,5β-THF), while microsomal incubations generate upto 7 metabolites, including 6β-hydroxycortisol (6β-OHF), and 6β-hydroxycortisone (6β-OHE), products of the cytochrome P450 (CYP) 3A subfamily. The aim of the present study was, therefore, to examine two of the main enzymes involved in cortisol metabolism, namely, microsomal 6β-hydroxylase and cytosolic 4-ene-reductase. In particular, we wished to assess the substrate specificity of these enzymes and identify compounds with inhibitory potential. Incubations for 30 min containing [³H]cortisol, potential inhibitors, microsomal or cytosolic protein (3 mg), and co-factors were followed by radiometric HPLC analysis. The K_m value for 6β-OHF and 6β-OHE formation was 15.2 ± 2.1 μM (mean ± SD; n = 4) and the V_max value 6.43 ± 0.45 pmol/min/mg microsomal protein. The most potent inhibitor of cortisol 6β-hydroxylase was ketoconazole (K_i = 0.9 ± 0.4 μM; N = 4), followed by gestodene (K_i = 5.6 ± 0.6 μM) and cyclosporine (K_i = 6.8 ± 1.4 μM). Both betamethasone and dexamethasone produced some inhibition (K_i = 31.3 and 54.5 μ, respectively). However, substrates for CYP2C (tolbutamide), CYP2D (quinidine), and CYP1A (theophylline) were essentially non-inhibitory. The K_m value for cortisol 4-ene-reductase was 26.5 ± 11.2 μM (n = 4) and the V_max value 107.7 ± 46.0 pmol/min/mg cytosolic protein. The most potent inhibitors were androstendione (K_i = 17.8 ± 3.3 μM) and gestodene (K_i = 23.8 ± 3.8 μM). Although both compounds have identical A-rings to cortisol, and undergo reduction, inhibition was non-competitive.     

Inhibition of antennal esterases of the egyptian armyworm Spodoptera littoralis by trifluoromethyl ketones

A series of aliphatic and aromatic trifluoromethyl ketones has been tested as inhibitors of the antennal esterases of the Egyptian armyworm Spodoptera littoralis, by evaluation of the extent of hydrolysis of [1-³H]-(Z,E)-9, 11-tetradecadienyl acetate (1), a tritiated analog of the major component of the sex pheromone. The most active compounds with a long chain aliphatic structure were 3-octylthio-1,1,1-trifluoropropan-2-one (2) (IC₅₀ 0.55 μM) and 1,1,1-trifluorotetradecan-2-one (4) (IC₅₀ 1.16 μM). The aromatic compounds were generally less potent inhbitors than the coressponding aromatic ones, although β-naphthyltrifuloromethyl ketone (10) exhibited a remarkable inhibitory activity (IC₅₀ 7.9 μM). Compounds 2, 4 and 10 exhibit a competitive inhibition with K_i values of 2.51×10⁻⁵ M, 2.98×10⁻⁵ M and 2.49×10⁻⁴ M, respectively. Some of the trifluoromethyl ketones tested were slow-binding inhibitors and compounds 2 and 10 are described as inhibitors of the antennal esterases of a moth for the first time.     

Properties of amino-terminal parathyroid hormone-related peptides modified at positions 11–13

Biological properties of amino-terminal PTHrP analogues modified in the region 11–13 were examined using ROS 17/2.8 cells. [Leu¹¹,D-Trp¹²,Arg¹³,Tyr³⁶]PTHrP(1–36)amide had a 17-fold lower binding affinity for the receptor (apparent K_d: 5 × 10⁻⁸ M) than [Tyr³⁶]PTHrP(1–36)amide or [Arg^11,13,Tyr³⁶]PTHrP(1–36)amide (apparent K_d for both: 2 × 10⁻⁹ M). Moreover, it is only a weak partial agonist despite completely inhibiting radioligand binding. [Leu¹¹,D-Trp¹²,Arg¹³,Tyr³⁶,Cys³⁸]PTHrP(7–38) and PTHrP(7–34)amide had similar receptor affinities (apparent K_ds: 5 × 10⁻⁸ M and 8 × 10⁻⁸ M), while that of [Nle^8,18,Tyr³⁴]bPTH(7–34)amide was more than 10-fold lower (apparent K_d: 2 × 10⁻⁶ M). These changes in biological properties suggest that high affinity receptor binding requires both amino- and carboxyl-terminal domains of the PTHrP(1–36) sequence and/or intramolecular interactions which are impaired by the D-Trp substitution for Gly¹².     

A performance comparison of choline biosensors: anodic or cathodic detections of H2O2 generated by enzyme immobilized on a conducting polymer

Amperometric choline biosensors were fabricated by the covalent immobilization of an enzyme of choline oxidase (ChO) and a bi-enzyme of ChO/horseradish peroxidase (ChO/HRP) onto poly-5,2′:5′,2″-terthiophene-3′-carboxylic acid (poly-TTCA) modified electrodes (CPMEs). A sensor modified with ChO utilized the oxidation process of enzymatically generated H₂O₂ in a choline solution at +0.6 V. The other one modified with ChO/HRP utilized the reduction process of H₂O₂ in a choline solution at −0.2 V. Experimental parameters affecting the sensitivity of sensors, such as pH, applied potential, and temperature were optimized. A performance comparison of two sensors showed that one based on ChO/HRP/CPME had a linear range from 1.0×10⁻⁶ to 8.0×10⁻⁵ M and the other based on ChO/CPME from 1.0×10⁻⁶ to 5.0×10⁻⁵ M. The detection limits for choline employing ChO/HRP/CPME and ChO/CPME were determined to be about 1.0×10⁻⁷ and 4.0×10⁻⁷ M, respectively. The response time of sensors was less than 5 s. Sensors showed good selectivity to interfering species. The long-term storage stability of the sensor based on ChO/HRP/CPME was longer than that based on ChO/CPME.     

Aromatase activity in the breast and other peripheral tissues and its therapeutic regulation

Studies using [³H]androstenedione (A) demonstrated that this substrate can be aromatized to estrone (E₁) in homogenates of breast carcinoma tissue and breast adipose tissue, in breast carcinoma and breast adipose stromal cells in culture, and in cultured adipose stromal cells from sites remote from the tumor. Using cultured breast carcinoma cells, it was shown that estrogen formation was stimulated by Cortisol (10⁻⁶ M) and inhibited by endogenous 5-reduced androgens: 5-androstene-dione>androsterone>dihydrotestosterone>epiandrosterone>3- and 3β-androstanediol. It was also shown that 19-nortestosterone and 19-norandrostenedione (10⁻⁶ M) inhibited E₁ formation by 80%. Progesterone (10⁻⁶ M) had no effect on aromatase activity, while the progestational agent R5020 (10⁻⁶ M) caused a 70% inhibition. These studies emphasize that a variety of compounds can influence aromatase activity and that drugs which are used as aromatase inhibitors in patients with breast carcinoma may have multiple sites of action.     

A nitrite reductase with cytochrome oxidase activity from Micrococcus denitrificans

The formation of nitrite reductase and cytochrome c in Micrococcus denitrificans was repressed by O₂. The purified nitrite reductase utilized reduced forms of cytochrome c, phenazine methosulphate, benzyl viologen and methyl viologen, respectively, as electron donors. The enzyme was inhibited by KCN, NaN₃ and NH₂OH each at 1 mM, whereas CO and bathocuproin, diethyl dithiocarbamate, o-phenanthroline and ,'-dipyridyl at 1 mM concentrations were relatively ineffective. The purified enzyme contains cytochromes, probably of the c and a₂ types, in one complex. A K_m of 46 μM for NO₂⁻ and a pH optimum of 6.7 were recorded for the enzyme. The molecular weight of the enzyme was estimated to be around 130000, and its anodic mobility was 6.8·10⁻⁶ cm²·sec⁻¹·V⁻¹ at pH 4.55.

The most highly purified nitrite reductase still exhibited cytochrome c oxidase activity with a K_m of 27 μM for O₂. This activity was also inhibited by KCN, NaN₃ and NH₂OH and by NO₂⁻.

A constitutive cytochrome oxidase associated with membranes was also isolated from cells grown anaerobically with NO₂⁻. It was inhibited by smaller amounts of KCN, NaN₃ and NH₂OH than the cytochrome oxidase activity of the nitrite reductase enzyme and also differed in having a pH optimum of about 8 and a K_m for O₂ of less than 0.1 μM. Spectroscopically, cytochromes b and c were found to be associated with the constitutive oxidase in the particulate preparation. Its activity was also inhibited by NO₂⁻.

The physiological role of the cytochrome oxidase activity associated with the purified nitrite reductase is likely to be of secondary importance for the following reasons: (a) it accounts for less than 10% of total cytochrome c oxidase activity of cell extracts; (b) the constitutive cytochrome c oxidase has a smaller K_m for O₂ and would therefore be expected to function more efficiently especially at low concentrations of O₂.