Ecdysone 20-Monooxygenase Activity of Cytochrome P450 in Ajuga reptans L. Plants and Cell Culture

The concentration of cytochrome P₄₅₀ and ecdysone 20-monooxygenase activity in plants and callus cell culture of carpet bugleweed Ajuga reptans L. were determined. The maximal ecdysone 20-monooxygenase activity of cytochrome P₄₅₀ was found in vegetative rosettes of intact plants. During the stage of flowering, the ecdysone 20-monooxygenase activity of cytochrome P₄₅₀ in plant leaves was higher than in other organs. It was demonstrated that the content of ecdysteroids in callus cell culture is higher than in the intact plant, with concurrent retention of a high ecdysone-20-monooxygenase activity.     

On the intracellular localization of the ecdysone 20-monooxygenase in Musca domestica. L

The cytochrome P-450-dependent 20-monooxygenation of ecdysone is catalyzed both by mitochondria and microsomes isolated from Musca domestica (L.) larvae; however, about 50% of the activity is associated with mitochondria, and 37% is associated with microsomes. Pretreatment of larvae with ecdysone results in an increase in Vmax and a decrease in Km values in mitochondria but not in microsomes. Phenobarbital, a known cytochrome P-450 inducer, increases the cytochrome P-450 levels in microsomes without affecting the 20-monooxygenase activity, but both the cytochrome P-450 levels and monooxygenase activity are depressed in mitochondria from phenobarbital-pretreated larvae. The ecdysone 20-monooxygenase activity is equally distributed between mitochondria and microsomes in adult insects. Pretreatment of the insects with ecdysone does not significantly modify the 20-monooxygenase activity of either mitochondrial or microsomal fractions, but the cytochrome P-450 levels are reduced in mitochondria. Phenobarbital also depresses the mitochondrial cytochrome P-450 levels while markedly increasing the microsomal cytochrome P-450 levels. However, no significant changes in ecdysone 20-monooxygenase activity are produced by phenobarbital pretreatment. The effects of ecdysone on adult cytochrome P-450 are mostly evidenced in mitochondria isolated from females, whereas in males the changes are not statistically significant. It is concluded that the mitochondrial ecdysone 20-monooxygenase is under regulatory control by ecdysone in the larval stage, which suggests that only the mitochondrial activity has a physiological role during insect development in M. domestica. In adults, both the mitochondrial and microsomal ecdysone 20-monooxygenase activities are not responsive to ecdysone, which, coupled to their high Km values, indicates that the reaction may not be of physiological importance in adult insects and that the mitochondrial cytochrome P-450 species being depressed by ecdysone in females are possibly not involved in ecdysone metabolism.     

Allelochemical stimulation of ecdysone 20-monooxygenase in fall armyworm larvae

The influence of dietary allelochemical on ecdysone 20-monooxygenase activity was studied in the fall armyworm, Spodoptera frugiperda (J.E. Smith). Feeding the indoles (indole-3-carbinol, indole-3-acetonitrile), flavonoids (flavone, β-naphthoflavone), monoterpenes (menthol, menthone, peppermint oil), and a coumarin (xanthotoxin) to the larvae stimulated midgut microsomal ecdysone 20-monooxygenase activity from 28 to 200% as compared with the controls. β-Naphthoflavone was the most potent inducer among those tested. Phenobarbital, a well-known cytochrome P450 inducer, also caused a 2-fold increase in the microsomal ecdysone 20-monooxygenase activity. Ecdysone 20-monooxygenase activity was 2.7-fold higher in the microsomal fraction than in the mitochondrial fraction isolated from larval midguts. Microsomal ecdysone 20-monooxygenase activity was highest in the fat body, followed by the midgut and Malpighian tubules. Tissue localization and enzyme inducibility were different between ecdysone 20-monooxygenase and xenobiotic-metabolizing cytochrome P450 monooxygenases, including aldrin epoxidase, biphenyl hydroxylase, methoxyresorufin O-demethylase, 7-ethoxycoumarin O-deethylase, p-chloro-N-methylaniline N-demethylase, and phorate sulfoxidase in fall armyworm larvae. © 1995 Wiley-Liss, Inc.     

Ecdysterone biosynthesis: a microsomal cytochrome-P-450-linked ecdysone 20-monooxygenase from tissues of the African migratory locust.

Ecdysone 20-monooxygenase, an enzyme which converts ecdysone to ecdysterone (the major moulting hormone of insects) has been characterized in cell-free preparations of tissues from African migratory locust. The product of the reaction has been identified as ecdysterone on the basis of several microchemical derivatization and chromatographic methods. Ecdysone 20-monooxygenase activity is located primarily in the microsomal fraction which also carries NADPH cytochrome c reductase and cytochrome P-450, as shown by sucrose density gradient centrifugation. Optimal conditions for the ecdysone 20-monooxygenase assay have been determined. The enzyme has a Km for ecdysone of 2.7 x 10(-7) M and is competitvely inhibited by ecdysterone (Ki = 7.5 x 10(-7) M). Ecdysone 20-monooxygenase is a typical cytochrome P-450 linked monooxygenase: the reaction requires O2 and is inhibited by CO, an effect partially reversed by white light. The enzyme is effectively inhibited by several specific monooxygenase inhibitors and by sulfhydryl reagents, but not by cyanide ions. Ecdysone elicits a type I difference spectrum when added to oxidized microsomes. NADPH acts as preferential electron donor. The transfer of reducing equivalents proceeds through NADPH cytochrome c (P-450) reductase: ecdysone 20-monooxygenase is inhibited by cytochrome c. Both NADPH cytochrome c reductase and ecdysone 20-monooxygenase are inhibited by NADP+ and show a similar Km for NADPH. The Malpighian tubules have the highest specific activity of ecdysone 20-monooxygenase, while fat body contain most of the cytochrome P-450 and NADPH cytochrome c reductase.     

A microsomal ecdysone-binding cytochrome P450 from the insect Locusta migratoria purified by sequential use of type-II and type-I ligands.

A dual-affinity method was established to purify, for the first time, a microsomal ecdysone-binding cytochrome P450 protein from locust Malpighian tubules. This method involved, after prepurification on omega-octylamino-agarose and hydroxylapatite, binding of cytochrome P450 to an immobilized triazole-based general P450 inhibitor (type-II ligand) followed by elution with the substrate ecdysone (type-I ligand) of the bound cytochrome. The isolated material showed a typical cytochrome P450 spectrum, a specific heme content of 13 nmol/mg protein, and a prominent protein of about 60 kDa on SDS-PAGE. Based on a tryptic undecapeptide sequence the isolated protein may be identical to CYP6H1, a putative ecdysone 20-monooxygenase recently cloned from the same tissue. Ecdysone 20-monooxygenase activity could be partially reconstituted from microsomal detergent extracts, when supplemented with purified bovine cytochrome P450 reductase and detergent-extracted microsomes; reconstitution was not successful with any chromatographic fraction, however. Therefore, purification of the locust cytochrome P450 was monitored by ecdysone-induced type-I difference spectra, whenever applicable, in addition to carbon monoxide spectra. Affinity columns with matrix-bound diethylstilbestrol and testosterone 3-thiosemicarbazone, but not with the 17beta-hemisuccinate, yielded elution profiles with ecdysone that were comparable to those of the triazole matrix. The concept of dual-affinity chromatography described here may be generally applicable to the isolation of cytochromes P450.     

The first cytochrome P450 in ferns. Evidence for its involvement in phytoecdysteroid biosynthesis in Polypodium vulgare

The fern Polypodium vulgare is a phytoecdysteroid (PE)-producing plant. Cultures of P. vulgare prothalus produce PE, whereas prothalus-derived callus cultures do not. However, this callus line can transform topically applied ecdysone (E) to 20-hydroxyecdysone (20E), which is the last step in the biosynthetic pathway of the main plant PE. This hydroxylation is catalysed by a cytochrome P450 enzyme. E treatment of the callus line results in an increased amount of P450, showing a linear correspondence between the amount of P450 and in vivo E 20-hydroxylation activity, estimated by measuring the bioconversion of E to 20E. This activity can be inhibited by molecules that bind to the P450-heme group. E shows a P450-substrate-binding spectrum with microsomes that overexpress the P450 protein. Finally, a P450 protein was purified from E-treated calli, this being the first P450 to be described in the pterydophyte group.     

Characterization of ecdysone 20-monooxygenase activity in wandering stage larvae of Drosophilamelanogaster: Evidence for mitochondrial and microsomal cytochrome P-450 dependent systems

Ecdysone 20-monooxygenase, the enzyme system that hydroxylates ecdysone to 20-hydroxyecdysone, was characterized in wandering stage larvae of Drosophila melanogaster using an in vitro radioassay in conjunction with analytical thin layer chromatography. 20-Hydroxyecdysone was confirmed to be the product of the enzyme radioassay system by high pressure liquid chromatography. The 20-monooxygenase was found to be most active in a 0.10 M phosphate buffer, pH 7.5, was inhibited by Ca²⁺, Mg²⁺ and Se⁴⁺ and exhibited a temperature optimum at 35°C. Differential centrifugation, sucrose step gradient centrifugation, electron microscopy and organelle-marker enzyme analysis revealed that ecdysone 20-monooxygenase activity is associated with both the mitochondrial and microsomal fractions. Substrate kinetics experiments indicated that the mitochondrial and microsomal monooxygenase systems exhibit apparent K_ms for ecdysone of 6.4 × 10⁻⁸ and 9.9 × 10⁻⁸ M, respectively, with apparent V_maxs of 4.1 and 10.2 pg 20-hydroxyecdysone formed/min per mg tissue equiv., respectively. Both monooxygenase systems were inhibited by their product 20-hydroxyecdysone. The cytochrome P-450 nature of these insect steroid hydoxylases was initially suggested by their requirement for NADPH, NADH was approximately half as effective in supporting the mitochrondrial monooxygenase activity. In addition, both monooxygenase systems were inhibited by carbon monoxide, ellipticine, p-chloromercuribenzoate, metyrapone and p-aminoglutethimide but not by cyanide. Photochemical action spectra of ecdysone 20-monooxygenase activity confirmed the cytochrome P-450 dependency of both the mitochondrial and microsomal ecdysone 20-hydroxylase systems.     

Regulation of cytochrome P-450 dependent steroid hydroxylase activity in Manduca sexta: effects of the ecdysone agonist RH 5849 on ecdysone 20-monooxygenase activity

The non-steroidal ecdysone agonist RH 5849 (1,2-dibenzoyl-1-tert-butylhydrazine) was found to inhibit in a dose-response and apparently competitive fashion the cytochrome P-450 dependent ecdysone 20-monooxygenase activity in the midgut of wandering stage last instar larvae of the tobacco hornworn, Manduca sexta. More effectively on a per molar basis than the naturally occurring molting hormones ecdysone and 20-hydroxyecdysone, RH 5849 was also found to elicit the dramatic 50-fold increase in midgut steroid hydroxylase activity (which normally occurs with the onset of the wandering stage) when injected into competent head or thoracic ligated pre-wandering last instar larvae. These data support and extend the potential usefulness of RH 5849 as a pharmacological probe for further investigating the actions of ecdysteroids and their role(s) in the regulation of ecdysteroid monooxygenases.     

Ethanol increases cytochromes P450IIE, IIB1/2, and IIIA in cultured rat hepatocytes

In intact rats, ethanol treatment has been associated with increases in hepatic levels of both P450IIB1/2 and P450IIE. When rat hepatocytes were cultured on an extracellular tumor matrix (Matrigel), exposure to ethanol from 48 to 96 h in culture resulted in increases in cytochromes P450IIE, IIB1/2, and IIIA. Cytochrome P450IIE was detected immunologically and enzymatically, using two activities associated with cytochrome P450IIE, p-nitrophenol hydroxylation, and acetaminophen activation to a metabolite that binds to glutathione. The content of cytochrome P450IIE in freshly isolated cells decreased when the cells were placed in culture. Exposure of the cultured hepatocytes to ethanol from 48 to 96 h after inoculation resulted in an increase in cytochrome P450IIE compared to untreated cultured cells. In addition, in culture, the amount of enzymatically active protein after ethanol treatment was equal to that in hepatocytes freshly isolated from intact animals. Ethanol treatment resulted in increases in cytochrome P450IIB1/2 compared to untreated cells, as shown immunologically and by increased benzyloxyresorufin dealkylase activity. However, phenobarbital induced cytochrome P450IIB1/2 to higher levels, compared to ethanol. Ethanol and phenobarbital treatments both increased P450IIIA, as determined immunologically and by the amount of propoxycoumarin depropylase activity that is inhibited by triacetyloleandomycin. However, the amount of P450IIIA increased after ethanol treatment was less than that increased after treatment with dexamethasone in these cells. The ethanol-mediated increases in all four forms of cytochrome P450 in culture suggest that these increases in the intact animal result from direct effects of ethanol on the liver.     

Ecdysone 20-monooxygenase activity in land crabs

1. Ecdysone 20-monooxygenase activity has been found in hepatopancreas, gonads, epidermis and muscle of the crab Gecarcinus lateralis. Activity was assayed by measuring the in vitro conversion of [³H]-ecdysone to [³H]-20-hydroxyecdysone. Maximal activity is obtained at 30°C and pH 8.0 in sodium phosphate buffer.2. Activity from hepatopancreas is localized in a fraction which sediments at 10,000 g, probably mitochondria.3. NADPH stimulates activity and metyrapone or oxygen deprivation inhibits it, as has been observed for cytochrome P-450-dependent monooxygenases.4. Changes in ecdysone 20-monooxygenase activity at different stages of the molt cycle are not directly correlated to changes in ecdysteroid levels in the hemolymph.     

Molecular cloning of NADPH-cytochrome P450 oxidoreductase from silkworm eggs. Its involvement in 20-hydroxyecdysone biosynthesis during embryonic development.

Using RT-PCR, a cDNA fragment of NADPH-cytochrome P450 oxidoreductase from silkworm, Bombyx mori, was cloned from three-day-old nondiapause eggs. RACE was used to isolate the ends of the DNA. The full-length cDNA obtained was composed of 3471 bp with an open reading frame encoding a protein of 687 amino-acid residues with a relative molecular mass of 77 700. The protein, fused with glutathione S-transferase, was expressed in Escherichia coli and purified to homogeneity. The fused protein not only had NADPH-dependent cytochrome c-reducing activity, but also acted as an electron carrier from NADPH to bovine adrenal 21-hydroxylase P450 in the steroid hydroxylation reaction, confirming that the protein is the silkworm NADPH-cytochrome P450 oxidoreductase. Ecdysone 20-hydroxylase activity in the nondiapause egg microsomes increased until the fourth day after oviposition, and then decreased, little being detected on the ninth day. An antibody raised against the P450 reductase inhibited the ecdysone hydroxylation. Immunoblot analyses of the microsomes indicated that the P450 reductase protein appeared distinctly in the three-day-old nondiapause eggs and, in contrast to the developmental pattern of ecdysone hydroxylase activity, continued to increase as the embryos developed. These results suggest that ecdysone hydroxylation in the early stage of embryogenesis is dependent on the presence of both P450 reductase and ecdysone 20-hydroxylase P450, but its gradual reduction in the later stage may be due to the decrease in the level of ecdysone 20-hydroxylase P450.     

Isolation of immunochemically distinct form of cytochrome P-450 from microsomes of tulip bulbs

A highly purified cytochrome P-450 was obtained from the microsomes of tulip bulbs (Tulipa gesneriana L.). The molecular weight (Mr = 52,500) and amino acid composition of this plant cytochrome P-450 are similar to those reported for rat livers. On the contrary, Ouchterlony double diffusion analyses indicated that cytochrome P-450 isolated from tulip bulbs shares no common antigenic determinants with those of 9 other plants, in spite of the presence of comparable contents of cytochrome P-450 and/or trans-cinnamate 4-monooxygenase with tulip bulbs.     

Effects of azadirachtin on insect cytochrome P-450 dependent ecdysone 20-monooxygenase activity

The effects of the insect growth and ecdysis inhibitor azadirachtin on ecdysone 20-monooxygenase activity were examined in three insect species. Homogenates of wandering stage third instar larvae of Drosophila melanogaster, or abdomens from adult female Aedes aegypti, or fat body or midgut from last instar larvae of Manduca sexta were incubated with radiolabelled ecdysone and increasing concentrations of azadirachtin and the ecdysone 20-monoxygenase activity quantified by radioassay. Azadirachtin was found to inhibit in a dose-response fashion the ecdysone 20-monooxygenase activity associated with all the insect preparations. The concentration of azadirachtin required to elicit approximately 50% inhibition of the ecdysone 20-monooxygenase activity ranged from a low of 1 x 10(-4) M for Drosophila to a high of 4 x 10(-4) M for Manduca midgut.     

Ecdysone 20-mono-oxygenase in the desert locust, Schistocerca gregaria.

The enzyme catalysing the hydroxylation of ecdysone to 20-hydroxyecdysone, ecdysone 20-mono-oxygenase (EC 1.14.99.22), was investigated in the Malpighian tubules of fifth-instar locusts, Schistocerca gregaria. Enzyme activity was optimal at 35 degrees C and pH 6.8-8.0. Under these conditions the mono-oxygenase exhibited an apparent Km for ecdysone of 7.1 X 10(-7) M, a maximal specific activity of 1.1 nmol/h per mg of protein and was competitively inhibited by 20-hydroxyecdysone with an apparent Ki of 6.3 X 10(-7) M. Enzyme activity was decreased in the presence of Ca2+, Mg2+, EDTA and non-ionic detergents. The Malpighian tubule ecdysone 20-mono-oxygenase was localized primarily in the subcellular fraction sedimenting at 7500 g and, on the basis of marker enzyme profiles, was assigned mainly to the mitochondria. NADPH was required for activity, although addition of NADH together with NADPH had a synergistic effect. NADP+-dependent isocitrate dehydrogenase (EC 1.1.1.42) and an energy-dependent NAD(P) transhydrogenase (EC 1.6.1.1.) appeared to be the major sources of reducing equivalents, with the contribution from the 'malic enzyme' (EC 1.1.1.40) being less important. The monooxygenase was characterized as a cytochrome P-450-containing mixed-function oxidase from the inhibition patterns with metyrapone, CO and cyanide; CO inhibition was reversible with monochromatic light at 450 nm. However, the ecdysone 20-mono-oxygenase shows much lower sensitivity to CO inhibition and to photodissociation of the CO-inhibited complex than do vertebrate cytochrome P-450-dependent hydroxylation systems. The concentration of cytochrome P-450 in the Malpighian tubule mitochondria was 30 pmol/mg of protein. The properties of the mono-oxygenase are discussed in relation to hydroxylation enzymes from other sources.     

The effect of alpha-ecdysone and phenobarbital on the alpha-ecdysone 20-monooxygenase of house fly larva

The NADPH-dependent cytochrome P-450 20-monooxygenation of alpha-ecdysone is catalyzed both by mitochondria and microsomes isolated from Musca domestica, L. larvae, but about 50% of the activity is associated with mitochondria and 37% with microsomes. The mitochondrial activity is increased by pretreatment with alpha-ecdysone with a concomitant decrease in Km values. This effect is not observed in microsomes. Induction with phenobarbital represses the mitochondrial 20-monooxygenase but does not change the microsomal activity, although a large increase in cytochrome P-450 is observed in the latter fraction. It is concluded that only the mitochondrial 20-monooxygenase appears to be regulated by alpha-ecdysone which suggests that mitochondrial cytochrome P-450 forms are involved in the moulting phenomenon; whereas, microsomal cytochrome P-450 activity may be of a nonspecific nature and not relevant to development.     

Apparent maintenance of cytochrome P 450 by nicotinamide in primary cultures of rat hepatocytes.

The sole addition of a high, unphysiological, concentration of nicotinamide (25 mM) to a cell culture medium was found to maintain the cytochrome P 450 concentration of rat hepatocytes cultured for 24 hours at 71% of the level found in intact liver, whilst hepatocytes cultured without nicotinamide contained only 20% of their initial cytochrome P 450. Furthermore the P 450 concentration of hepatocytes cultured for 24 hours in the presence of 25 mM nicotinamide could be increased to the same level as found in intact rat liver by the inclusion of 1 mM nicotinamide into the medium used for cell isolation. Although the mechanism of action of nicotinamide is unknown this simple system for the maintenance of cytochrome P 450 in hepatocyte culture could provide the opportunity to study, under defined conditions $\text{in vitro}$, the factors that regulate cytochrome P 450 and hence determine hepatotoxicity and hepatocarcinogenesis.     

Possible role for covalent modification in the reversible activation of ecdysone 20-monooxygenase activity

Evidence is presented for the reversible activation-inactivation of the microsomal ecdysone 20-monooxygenase from fat body of the cotton leafworm, Spodoptera littoralis, in a manner commensurate with reversible changes in its phosphorylation state. The activity of the monooxygenase was higher following preincubation with fluoride (an inhibitor of phosphoprotein phosphatases) than in its absence. Preincubation with alkaline phosphatase or with cAMP-dependent protein kinase resulted in appreciable diminution or enhancement, respectively, in monooxygenase activity. Activation of ecdysone 20-monooxygenase activity could also be effected by incubation with a cytosolic fraction in the presence of cAMP, ATP, and fluoride; this activation was prevented by a cAMP-dependent protein kinase inhibitor. Similarly, inactivation of the monooxygenase was achieved by preincubation with cytosol, the effect being enhanced by Ca²⁺-calmodulin or by Mg²⁺ ions. The combined results provide indirect evidence that the microsomal ecdysone 20-monooxygenase exists in an active phosphorylated form and an inactive dephosphorylated form, interconvertible by a cAMP-dependent protein kinase and a phosphoprotein phosphatase.     

Regulation of ecdysone hydroxylation in Locusta migratoria: Rôle of the moulting hormone level

After repetitive injections of moderate doses of ecdysone, ecdysterone or phenobarbital to young Vth (last) instar larvae of Locusta migratoria, the conversion rate of ecdysone to ecdysterone in vivo is significantly higher than in control insects. Similarly, 5 hr after injection of a low dose of ecdysone or ecdysterone, a strong ‘induction’ of ecdysone 20-monooxygenase activity occurs. This ‘inductive’ effect is blocked by cycloheximide.Simultaneous injections of ecdysone and ecdysterone show that hydroxylation of ecdysone is inhibited by the product of the reaction, ecdysterone. Removal of the prothoracic glands and X-ray treatment of the hemocytopoietic tissue do not affect ecdysone hydroxylation. The mechanism of induction and inhibition of ecdysone 20-monooxygenase shown in this study is probably responsible for the important variations of this key enzyme which have been reported from several insect species.     

Ecdysone 20-monooxygenase activity in Drosophila virilis strains varying in ecdysteroid response to heat stress

The effect of various duration of heat stress (38 degrees C) on the activity of ecdysone 20-monooxygenase converting ecdysone into 20-hydroxyecdysone has been studied in D. virilis of wild type and mutant strain females, which differ by the mode of heat stress response of ecdysone and 20-hydroxyecdysone. We are the first to show that heat stress induces activity of ecdysone 20-monooxygenase in Drosophila females and enzyme activity correlates with the level of 20-hydroxyecdysone.