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1.
Androst-4-ene-3,17-dione (androstenedione) was found to be a potent competitive inhibitor of the NADH-supported reduction of retinal in rat hepatic microsomes ( Ki 42 μM, Km/ Ki ratio 1.1). Similarly, the NADH-mediated reduction of androstenedione was inhibited in mixed fashion by retinal ( Ki 12 μM, Km/ Ki ratio 0.34). In subsequent experiments the cofactor NADH exhibited an identical Km (8 μM) in the microsomal reductions of both substrates. Acidic pH markedly stimulated the microsomal reduction of androstenedione to testosterone and was also found to enhance retinal reduction to retinol, although the latter reaction exhibited a district pH optimum between 6.0 and 6.5. These results suggest that a common enzyme may participate in the reduction of both substrates but at least one other enzyme probably participates in hepatic microsomal testosterone production. 相似文献
2.
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 Km value for 6β-OHF and 6β-OHE formation was 15.2 ± 2.1 μM (mean ± SD; n = 4) and the Vmax value 6.43 ± 0.45 pmol/min/mg microsomal protein. The most potent inhibitor of cortisol 6β-hydroxylase was ketoconazole ( Ki = 0.9 ± 0.4 μM; N = 4), followed by gestodene ( Ki = 5.6 ± 0.6 μM) and cyclosporine ( Ki = 6.8 ± 1.4 μM). Both betamethasone and dexamethasone produced some inhibition ( Ki = 31.3 and 54.5 μ, respectively). However, substrates for CYP2C (tolbutamide), CYP2D (quinidine), and CYP1A (theophylline) were essentially non-inhibitory. The Km value for cortisol 4-ene-reductase was 26.5 ± 11.2 μM ( n = 4) and the Vmax value 107.7 ± 46.0 pmol/min/mg cytosolic protein. The most potent inhibitors were androstendione ( Ki = 17.8 ± 3.3 μM) and gestodene ( Ki = 23.8 ± 3.8 μM). Although both compounds have identical A-rings to cortisol, and undergo reduction, inhibition was non-competitive. 相似文献
3.
The microsomal fraction from the testes of immature pigs (<1 week old) contains 3β-hydroxysteroid dehydrogenase-isomerase (3β-HSD-isomerase) activities that convert dehydroepiandrosterone (DHA) to 4-androstenedione and 5,16-androstadien-3β-ol (andien-β) to 4,16-androstadien-3-one (dienone). These reactions are necessary for the biosynthesis of hormonally and pheromonally active steroids. Kinetic analyses of these activities were done to determine whether they are catalysed by a single enzyme or if there is any interaction between the substrates and products of one reaction on the activity of the other enzyme. Kinetic parameters were determined and the affinities for steroid substrate were similar (7–9 μmol/l) but the Vmaxapp value for the conversion of andien-β to dienone was 10-fold that of the DHA to 4-androstenedione reaction. In analyses of the conversion of DHA to 4-androstenedione, neither andien-β nor dienone inhibited the reaction and especially, no effect on the Kmapp for DHA was observed which would have indicated competition between DHA and andien-β for the same active site ( Kiapp from slope and intercept replots were between 3 and 80 times the values of the kinetic constants). Similarly, DHA and 4-androstenedione had minor or negligible effects on the conversion of andien-β to dienone ( Kiapp from slope replots were the same as the Kmapp but the Kiapp from the intercept replot was 12 to 25% of the Vmaxapp). It is concluded that substrate specific 3β-HSD-isomerases for andien-β and DHA exist in the immature pig testis and there is little, if any interaction between these enzymes. 相似文献
4.
In the adult rat, the duodenal tissue of both sexes can convert progesterone to 17-hydroxyprogesterone, androstenedione and testosterone. The transition from C 21 to C 19 steroids is apparently controlled by the same cytochrome P450c17 expressed in the testis, which catalyzes both 17-hydroxylation and C-17,20 bond scission at a single bifunctional active site. The kinetic parameters of this enzyme were measured at the steady state for both reactions using [1,2- 3H]progesterone and [1,2- 3H]17-hydroxyprogesterone as substrates. In the testis and male and female duodena, the Km values for progesterone 17-hydroxylation were 14.2, 23.8 and 23.2 nM, whereas the Vmax values were 105, 3.5 and 3.1 pmol/mg protein/min, respectively. With respect to C-17,20 lyase activity, the Km values for exogenous 17-hydroxyprogesterone were 525, 675 and 637 nM, whereas the Vmax values were 283, 7.8 and 7.8 pmol/mg protein/min, respectively. However, when the Km values were calculated with respect to intermediate 17-hydroxyprogesterone formed from progesterone, they were similar to the Km values for 17-hydroxylase, being 15, 31.4 and 24.8 nM, whereas the Vmax values were 26.3, 2 and 1.8 pmol/mg protein/min, respectively. The similarity of Km values is due to the fact that the relative androgen formation efficiency (bond scission events/total 17-hydroxylation events ratio) was remarkably constant in both testicular and duodenal incubates, irrespective of progesterone concentration. Efficiency values were 2-fold higher in duodenal tissue (0.54) than in testis (0.25). Estradiol-17β inhibited 17-hydroxylation but not bond scission on intermediate 17-hydroxyprogesterone, because it did not affect the efficiency value. Rat duodenal P450c17 has the same substrate affinity, a lower specific activity and a higher androgen formation efficiency than testicular P450c17. 相似文献
5.
1. 1. Esterification of 32P1 by illuminated chloroplasts prepared on a sucrose gradient was examined to establish the optimal incubation conditions. 2. 2. The evidence is consistent with phosphorylation being closely coupled to the sum of noncyclic and pseudocyclic electron flow and with the rate of electron flow responding to the availability of electron acceptors. 3. 3. Apparent Km values for ADP and Mg2+ were found to be 40 and 250 μM, respectively. The Km value for Mg2+ was increased by the presence of Ca2+. Two apparent values were observed for P1 at 0.2 and 1.1 mM. Chloroplast damage resulted in increased apparent Km (P1) values. 4. 4. Acceleration of the esterification resulting from the addition of ADP and P1 to the medium indicated that these compounds were able to penetrate to the active site of esterification. 5. 5. Ribose 5-phosphate (Rib-5-P) was shown to inhibit P1 esterification without affecting the apparent Km for ADP or P1. The evidence suggests that Rib-5-P interferes with the uptake of P1, and possibly ADP.
Abbreviations: PMS, phenazine methosulphate; CMU, 1-(p-chlorophenyl)-3,3′-dimethylurea 相似文献
6.
1. The membrane bound DPNH oxidase of Escherichia coli can reduce the artificial electron acceptors: ferricyanide, dichlorophenolindophenol (DCIP) and menadione. All three are reduced by the flavoprotein of DPNH oxidase, but at different sites of the enzyme. 2. Freeze-drying of the bacterial membranes causes a selective detachment of DPNH dehydrogenase (DPNH: (acceptor) oxidoreductase, EC 1.6.99.3) from the membranes. This solubilization is accompanied by a decrease of Km(K3Fe(CN)6) from 2.0 to 0.25 mM, while no change is detected in Km(DPNH). This enzyme is not the DPNH diaphorase found in the bacteria. 3. DPNH dehydrogenase of E. coli is a metalloflavoprotein, containing non-heme iron, labile sulfide, FMN and FAD. 4. Reduction of the enzyme with DPNH in the absence of electron acceptor (ferricyanide or DCIP) causes a rapid and irreversible change to a less active state, Form II. Form II is characterized by a higher Km(DPNH) and slower vmax., while the Km(K3Fe(CN)6) remains unchanged. 5. The transformation of the enzyme to Form II is accompanied by the reduction of the non-heme iron component. The role of non-heme iron in the enzymic reaction is discussed. 相似文献
7.
In order to better understand the function of aromatase, we carried out kinetic analyses to asses the ability of natural estrogens, estrone (E 1), estradiol (E 2), 16-OHE 1, and estriol (E 3), to inhibit aromatization. Human placental microsomes (50 μg protein) were incubated for 5 min at 37°C with [1β- 3H]testosterone (1.24 × 10 3 dpm 3H/ng, 35–150 nM) or [1β- 3H,4- 14C]androstenedione (3.05 × 10 3 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 Ki of E 1, E 2, 16-OHE 1, and E 3 for testosterone aromatization was 1.5, 2.2, 95, and 162 μM, respectively, where the Km of aromatase was 61.8 ± 2.0 nM ( n = 5) for testosterone. The Ki of E 1, E 2, 16-OHE 1, and E 3 for androstenedione aromatization was 10.6, 5.5, 252, and 1182 μM, respectively, where the Km 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. 相似文献
8.
The formation of nitrite reductase and cytochrome c in Micrococcus denitrificans was repressed by O 2. 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, NaN 3 and NH 2OH 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 Km of 46 μM for NO 2− 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 cm 2·sec −1·V −1 at pH 4.55. The most highly purified nitrite reductase still exhibited cytochrome c oxidase activity with a Km 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 Km 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 Km for O2 and would therefore be expected to function more efficiently especially at low concentrations of O2. 相似文献
9.
The effect of organic solvents on carboxypeptidase Y (a serine carboxypeptidase from yeast)-catalyzed hydrolysis of amino acid ester and peptide synthesis from N-acyl amino acid ester and amino acid amide was investigated.
The Km value of ester hydrolysis increased with an increase in the solvent content. Dioxane was the most effective and dimethyl sulfoxide (DMSO) the least, whilst Kcat showed a tendency to increase slightly in N, N-dimethylformamide (DMF) and DMSO. For dioxane and acetonitrile (MeCN) a maximum was observed.
In peptide formation from Fua-Phe-OEt and Gly-NH 2, dioxane and MeCN supported high product yield at molar fractions smaller than ca. 0.05 but the yield decreased significantly at higher fractions, although a relatively constant selectivity (ratio of the peptide bond formed to the ester consumed) was maintained. DMSO gave rather low peptide yields and selectivity even at lower molar fractions. DMF showed an intermediate tendency.
An apparent saturation parameter of the amine component was evaluated and the dissociation constant of a complex between acyl-enzyme and amino acid amide ( Kn), as well as the rate constant of aminolysis exerted by the amino acid amide bound correctly on the enzyme ( Kn), was calculated by initial rate analysis of peptide formation. In contrast to Km values, Kn decreased with increasing concentrations of organic cosolvent. while a suppressive effect was observed (except for DMSO) on the Kn parameter.
Effects of the solvent practically immiscible in water was also studied by use of the enzyme physically “immobilized” on glass beads. 相似文献
10.
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 Km of 1.58 μM 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 μM, respectively, for testosterone aromatization, and 5.0 and 8.1 μM, respectively, for androstenedione aromatization. Androstenedione and testosterone showed Ki 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. 相似文献
11.
ATPase activity of photosynthetic membrane fragments from the bacterium Rhodopseudomonas capsulata can be stimulated by continuous illumination under conditions of active cyclic electron flow. The activation corresponds to an increase in the maximum velocity of the reaction and does not affect the apparent Km for ATP (0.11 mM). No stimulation in the light is observed in the presence of classical uncouplers or oxidized 2,6-dichlorophenolindophenol (DCIP), which, per se, stimulate ATPase in the dark. It is demonstrated, however, that oxidized DCIP acts as an uncoupler of bacterial photophosphorylation. The effect of light is elicited after a few minutes of preillumination, or in a much shorter time if an ADP trapping system is supplied. Activation does not occur if ADP is added during the preillumination (apparent Km for inhibition by ADP = 1 μM). The effect of ADP is not related to competitive inhibition with ATP, which can be observed at higher concentrations (apparent Ki = 0.26 mM). ADP, however, is not effective if added after some minutes of preillumination. 相似文献
12.
The selective monoamine oxidase inhibitors clorgyline and (−)-deprenyl were used to study the distribution of monoamine oxidase-A and -B (MAO-A, MAO-B) activities towards (−)-noradrenaline and (+),(−)-adrenaline in homogenates from seven different regions of human brain. The activities towards 5-hydroxytryptamine and 2-phenethylamine, which are essentially specific substrates for the A- and B-forms, respectively, under the conditions used in this work, were also determined. Noradreanline and adrenaline were substrates for both forms of the enzyme in all regions studied. The total MAO activity was found to be highest in the hypothalamus and lowest in the cerebellar cortex. Use of the selective MAO inhibitors clorgyline and (−)-deprenyl also showed adrenaline and noradrenaline to be substrates for both forms of the enzyme in rat brain. In human cerebral cortex and rat brain the two forms were found to have similar Km-values and maximum velocities towards adrenaline. These values for the two forms were also found to be similar in human cerebral cortex when noradrenaline was used as the substrate. In contrast MAO-A showed a significantly lower Km and a higher maximum velocity towards noradrenaline in rat brain. These results suggest that the rat may not provide a close model of the human for studies on the effects of MAO inhibitors on brain noradrenaline metabolism. 相似文献
14.
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 (E 1 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 Km and Vmax of E 1S were determined to be 8.1 μM and 43 nmol/h/mg protein. The corresponding Ki 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. 相似文献
15.
An extracellular β-xylosidase from the thermophilic fungus Paecilomyces thermophila J18 was purified 31.9-fold to homogeneity with a recovery yield of 2.27% from the cell-free culture supernatant. It appeared as a single protein band on SDS–PAGE with a molecular mass of approx 53.5 kDa. The molecular mass of β-xylosidase was 51.8 kDa determined by Superdex 75 gel filtration. The enzyme was a glycoprotein with a carbohydrate content of 61.5%. It exhibited an optimal activity at 55 °C and pH 6.5, respectively. The enzyme was stable in the range of pH 6.0–9.0 and at 55 °C. The purified enzyme hydrolyzed xylobiose and higher xylooligosaccharides but was inactive against xylan substrates. It released xylose from xylooligosaccharides with a degree of polymerization ranging between 2 and 5. The rate of xylose released from xylooligosaccharides by the purified enzyme increased with increasing chain length. It had a Km of 4.3 mM for p-nitrophenol-β-d-xylopyranoside and was competitively inhibited by xylose with a Ki value of 139 mM. Release of reducing sugars from xylans by a purified xylanase produced by the same organism increased markedly in the presence of β-xylosidase. During 24-hour hydrolysis, the amounts of reducing sugar released in the presence of added β-xylosidase were about 1.5–1.73 times that of the reaction employing the xylanase alone. This is the first report on the purification and characterization of a β-xylosidase from Paecilomyces thermophila. 相似文献
16.
To study mechanisms of aromatase inhibition in brain cells, a highly effective non-steroidal aromatase inhibitor (Fadrozole; 4-[5,6,7,8-tetra-hydroimidazo-(1,5- a)-pyridin-5-yl] benzonitrile HCl; CGS 16949A) was compared with endogenous C-19 steroids, known to be formed in the preoptic area, which inhibit oestrogen formation. Using a sensitive in vitro tritiated water assay for aromatase activity in avian (dove) preoptic tissue, the order of potency, with testosterone as substrate was: Fadrozole ( Ki < 1 × 10 −9 M) > 4-androstenedione 5-androstanedione > 5-dihydrotestosterone ( Ki = 6 × 10 −8 M) > 5β-androstanedione > 5β-dihydrotestosterone ( Ki = 3.5 × 10 −7 M) > 5-androstane-3, 17β-diol ( Ki = 5 × 10 −6 M) > 5β-androstane-3β,17β-diol. Five other steroids, 5β-androstane-3,17β-diol, 5-androstane-3β,17β-diol, progesterone, oestradiol and oestrone, showed no inhibition at 10 −4 M. The kinetics indicate that endogenous C-19 steroids show similar competitive inhibition of the aromatase as Fadrozole. Mouse (BALB/c) preoptic aromatase was also inhibited by Fadrozole. We conclude that endogenous C-19 metabolites of testosterone are effective inhibitors of the brain aromatase, and suggest that they bind competitively at the same active site as Fadrozole. 相似文献
17.
1. 1.|No obvious correlation was found between the temperature dependence of the apparent Km values for either mitochondrial succinoxidase or NADH oxidase systems prepared from rat or rainbow trout liver tissue and the previously observed pattern of susceptibility of these same enzymes to thermal inactivation. 2. 2.|Apparent activation energies (EA) have been calculated from the data and, although these are equivocal when considered in the above context, it is concluded that the possibility is not excluded that entropy may be a more important variable than enthalpy in the action of these enzymes in ectothermic animals.
Author Keywords: Mitochondria; Salmo gairdneri; rat; succinoxidase; NADH oxidase; temperature dependence; Km; activation energy; enzyme evolution 相似文献
18.
The synthetic decapeptide H-SLTCLVKGFY-OH (termed immunorphin) corresponding to the sequence 364–373 of the CH3 domain of human immunoglobulin G heavy chain was found to compete with [ 125I]β-endorphin for high-affinity receptors on T lymphocytes from the blood of healthy donors ( Ki = 0.6 nM). Besides immunorphin, its synthetic fragments H-Val-Lys-Gly-Phe-Tyr-OH ( Ki = 15 nM), H-Leu-Val-Lys-Gly-Phe-Tyr-OH ( Ki = 8.0 nM), H-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH ( Ki = 3.4 nM), H-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH ( Ki = 2.2 nM), H-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH ( Ki = 1.0 nM) possessed the ability to inhibit specific binding of [ 125I]β-endorphin to T lymphocytes. Tests of the specificity of the receptors revealed that they are not sensitive to naloxone and Met-enkephalin, i.e. they are not opioid receptors. Kd values characterizing the specific binding of 125I- labeled immunorphin and its fragment H-Val-Lys-Gly-Phe-Tyr-OH to the receptors have been determined to be 7.4 nM and 36.3 nM, respectively. 相似文献
19.
Rates of microsomal 17β-estradiol (E 2) hydroxylation at the C-2, -4, -6, and -15 positions are each induced greater than 10-fold by treating MCF-7 breast cancer cells with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). The TCDD-induced activities at the C-2, -6 and -15 positions have been attributed to cytochrome P450 1A1 (CYP1A1); however, the low Km 4-hydroxylase induced by TCDD appears to be a distinct enzyme. We report here that antibodies to cytochrome P450-EF (mouse CYP1B1) selectivity inhibited the C-4 hydroxylation of E 2 catalyzed by microsomes from TCDD-treated MCF-7 cells. Western blots probed with anti-CYP1B antibodies showed the induction of a 52 kDa microsomal protein in response to treatment with TCDD in MCF-7 cells. Western blots of microsomes from HepG2 cells did not show the TCDD-induced 52 kDa protein, and microsomes from TCDD-treated HepG2 cells did not catalyze a low Km hydroxylation of E 2 at C-4. Cellular metabolism experiments also showed induction of both the C-2 and -4 hydroxylation pathways in TCDD-treated MCF-7 cells as evidenced by elevated 2- and 4-methoxyestradiol (MeOE 2) formation. In contrast, TCDD-treated HepG2 cells showed 2-MeOE 2 formation predominantly over 4-MeOE 2. Northern blots of RNA isolated from untreated and TCDD-treated cells, when probed with the human CYP1B1 cDNA, showed induction of a 5.2 kb RNA in MCF-7 cells but not in HepG2 cells in response to treatment with TCDD. These results provide additional evidence for the induction by TCDD of a novel E 2 4-hydroxylase in MCF-7 cells but not in HepG2 cells and indicate possible endocrine regulatory roles for the newly discovered group of enzymes of the CYP1B subfamily. 相似文献
20.
The endocervical mucosa is believed to be a primary site of HIV transmission. However, to date there is little known about the distribution of the HIV co-receptor CCR5 and the HIV-binding C-type lectin receptors, including Langerin, dendritic cell (DC)-specific intercellular adhesion molecule-grabbing non-integrin (DC-SIGN) and mannose receptor (MR) at this site. We therefore characterized the expression of these molecules in the endocervix of HIV seronegative women by computerized image analysis. Endocervical tissue biopsies were collected from women (n = 6) undergoing hysterectomy. All study individuals were diagnosed with benign and non-inflammatory diseases. CCR5+ CD4+ CD3+ T cells were found within or adjacent to the endocervical epithelium. The C-type lectin Langerin was expressed by intraepithelial CD1a+ CD4+ and CD11c+ CD4+ Langerhans cells, whereas DC-SIGN+ MR+ CD11c myeloid dendritic cells and MR+ CD68+ macrophages were localized in the submucosa of the endocervix. The previously defined immune effector cells including CD8+, CD56+, CD19+ and IgD+ cells were also found in the submucosa as well as occasional CD123+ BDCA-2+ plasmacytoid dendritic cells. Understanding the spatial distribution of potential HIV target cells and immune effector cells in relation to the endocervical canal forms a basis for deciphering the routes of HIV transmission events in humans as well as designing HIV-inhibiting compounds. 相似文献
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