Ecdysone 20-monooxygenase Activity Research Articles

Effects of Quinizarin and Five Synthesized Derivatives on Fifth Larval Instar Midgut Ecdysone 20-Monooxygenase Activity of the Tobacco HornwormManduca sexta

The plant allelochemical, quinizarin (1,4-dihydroxy-9,10-anthraquinone), and five anthraquinones that were synthesized from quinizarin, namely, 1,4-anthraquinone; 2-hydroxy-1,4-anthraquinone; 2-methoxy-1,4-anthraquinone; 9-hydroxy-1,4-anthraquinone; and 9-methoxy-1,4-anthraquinone, were assessed as to their effects on the essential, P450-dependent ecdysone 20-monooxygenase system of the insect modelManduca sexta(tobacco hornworm). This steroid hydroxylase converts the arthropod molting hormone, ecdysone, to the physiologically required 20-hydroxyecdysone form.M. sextafifth larval instar midgut homogenates were incubated with increasing concentrations (10−8to 10−3 M) of each of the six anthraquinones followed by ecdysone 20-monooxygenase assessments using a radioenzymological assay. Four of the five anthraquinones exhibitedI50’s of about4×10-6to6×10-2 M. The most effective inhibitors were 2-methoxy-1,4-anthraquinone and 1,4-anthraquinone followed by 9-hydroxy-1,4 anthraquinone and 9-methoxy-1,4-anthraquinone. At lower concentrations the latter anthraquinone stimulated E20M activity. Quinizarin was less inhibitory and 2-hydroxy-1,4-anthraquinone was essentially without effect. Significantly, these studies make evident for the first time that anthraquinones can affect insect E20M activity, and thus insect endocrine regulation and development, and that a relationship between anthraquinone structure and effectiveness is apparent. These studies represent the first demonstrations of anthraquinones affecting any steroid hydroxylase system.

Cyclic 3′, 5′ guanosine monophosphate and larval midgut ecdysone 20-monooxygenase activity of the tobacco hornworm, Manduca sexta

During the last larval stadium of the tobacco hornworm, Manduca sexta, midgut ecdysone 20-monooxygenase (E20M) activity increases 50-fold coincident with wandering onset. In our study, the effects of the ecdysone agonist RH-5849 and second messengers, dibutyryl cyclic 3', 5' adenosine monophosphate (dibutyryl cAMP) and dibutyryl cyclic 3', 5' guanosine monophosphate (dibutyryl cGMP), either alone or combined with other additives, on midgut E20M were assessed in vitro and in vivo. Pre-incubation of midguts with dibutyryl cAMP was ineffective, but dibutyryl cGMP elevated E20M activity. In vivo, the 50-fold increase in E20M activity was diminished by injections of guanylate cyclase inhibitors alone or combined with RH-5849. Guanylate cyclase inhibitor effects were significantly diminished when co-injected with dibutyryl cGMP, or a guanylate cyclase activator. Although dibutyryl cGMP injections into head- or thorax-ligated pre-wandering larvae did not elevate midgut E20M activity, they did synergize when co-injected with a suboptimal dose of RH-5849, thereby eliciting significant activity increases. Radioimmunoassays of midgut cGMP content throughout the M. sexta fifth larval instar revealed that levels of cGMP significantly increased between days four and five consistent with the 50-fold E20M activity increase. These data indicate cGMP involvement in the ecdysone-mediated increase in midgut E20M activity.

Midgut and fatbody mitochondrial transhydrogenase activities during larval-pupal development of the tobacco hornworm, Manduca sexta

Midgut and fatbody mitochondria from fifth larval instar Manduca sexta display a membrane-associated transhydrogenase that catalyzes a reversible hydride ion transfer between NADP(H) and NAD(H). The NADPH-forming activity occurs as a nonenergy- or energy-linked activity with energy for the latter derived from either electron transport-dependent NADH or succinate utilization, or ATP hydrolysis by Mg ++-dependent ATPase. During the ten-day developmental period preceding the larval-pupal molt (fifth larval instar), significant peaks in the mitochondrial transhydrogenase activities of midgut and fatbody tissues were noted and these peaks were coincident with the onset of wandering behavior and with the fifty-fold increase in ecdysone 20-monooxygenase (E20-M) activity previously reported for M. sexta midgut. Since E20-M preferentially uses NADPH in catalyzing ecdysone conversion to the physiologically active molting hormone, 20-hydroxyecdysone, the physiological and developmental significance of the mitochondrial, NADPH-forming energy-linked transhydrogenations were made apparent. Moreover, that the increases in all transhydrogenase activities resulted from de novo enzyme synthesis were indicated by the cycloheximide-dependent reductions in these activities.

Role of ecdysone 20-monooxygenase in regulation of 20-hydroxyecdysone levels by juvenile hormone and biogenic amines in Drosophila

The effects of increased levels of dopamine (feeding flies with dopamine precursor, L: -dihydroxyphenylalanine) and octopamine (feeding flies with octopamine) on ecdysone 20-monooxygenase activity in young (2 days old) wild type females (the strain wt) of Drosophila virilis have been studied. L: -dihydroxyphenylalanine and octopamine feeding increases ecdysone 20-monooxygenase activity by a factor of 1.6 and 1.7, respectively. Ecdysone 20-monooxygenase activity in the young (1 day old) octopamineless females of the strain Tbetah ( nM18 ), in females of the strain P845 (precursor of Tbetah ( nM18 ) strain) and in wild type females (Canton S) of Drosophila melanogaster have been measured. The absence of octopamine leads to a considerable decrease in the enzyme activity. We have also studied the effects of juvenile hormone application on ecdysone 20-monooxygenase activity in 2-day-old wt females of D. virilis and demonstrated that an increase in juvenile hormone titre leads to an increase in the enzyme activity. We discuss the supposition that ecdysone 20-monooxygenase occupies a key position in the regulation of 20-hydroxyecdysone titre under the conditions that lead to changes in juvenile hormone titre and biogenic amine levels.

Effects of the compounds 2‐methoxynaphthoquinone, 2‐propoxynaphthoquinone, and 2‐isopropoxynaphthoquinone on ecdysone 20‐monooxygenase activity

The effects of the natural compound 2-methoxy-1,4-naphthoquinone, isolated from the leaves of Impatiens glandulifera and the synthetic compounds 2-propoxy-1,4-naphthoquinone and 2-isopropoxy-1,4-naphthoquinone on ecdysone 20-monooxygenase (E-20-M) 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 fifth instar larvae of Manduca sexta were incubated with radiolabelled ecdysone and increasing concentrations (from 1 x 10(-8) to 1 x 10(-3) M) of the three compounds. All three compounds were found to inhibit in a dose-dependent fashion the E-20-M activity in the three insect species. The concentration of these compounds required to elicit a 50% inhibition of this steroid hydroxylase activity in the three insect species examined ranged from approximately 3 x 10(-5) to 7 x 10(-4) M.

Effect of octopamine on ecdysone-20 monooxygenase activity in Drosophila

Effect of octopamine on ecdysone-20 monooxygenase activity in Drosophila

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.

Ecdysone 20-Monooxygenase Activity of Cytochrome P450in 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.

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.

Ecdysone 20-monooxygenase activity during larval-pupal-adult development of the tobacco hornworm,Manduca sexta

Ecdysone 20-monooxygenase is the insect cytochrome P-450 dependent steroid hydroxylase system responsible for the conversion of ecdysone to 20-hydroxyecdysone. Using an in vitro radioassay, the levels of ecdysone 20-monooxygenase activity were quantified during embryogenesis in the egg, and in the fat body and midgut tissues during the fourth larval, fifth larval, and pupal-pharate adult stadia of the tobacco hornworm Manduca sexta. Although basal, ecdysone 20-monooxygenase activity was detected during the latter stages of larval embryogenesis. Postembryologically, a single major peak or increase was detected in both fat body and midgut tissues during each stadium. In the fourth larval instar, both fat body and midgut ecdysone 20-monooxygenase activities were found to peak in the latter half of the stadium at the time of spiracle apolysis; both activities dropped to basal levels by the time of ecdysis. During the fifth larval stadium, fat body steroid hydroxylase activity was found to rise during the feeding stage (days 1–4), then dropped back to basal levels by the onset of pharate pupal development on day 7 of the stadium. Midgut monoooxygenase activity during the fifth larval stadium, however, remained basal during the feeding stage, then rose dramatically to peak on day 5 with the onset of wandering, then dropped back to basal levels by the onset of pharate pupal development. Both fat body and midgut hydroxylase activities were basal during the pupal stage and only rose late in pharate-adult development, just prior to adult eclosion. Arch. Insect Biochem. Physiol. 41:24–32, 1999. © 1999 Wiley-Liss, Inc.

Ecdysone 20-monooxygenase in eggs of the silkworm, Bombyx mori: enzymatic properties and developmental changes.

Ecdysone 20-monooxygenase in eggs of the silkworm Bombyx mori was characterized in relation to embryonic development. First, subcellular fractions were prepared by means of differential centrifugation, and analyzed using marker enzymes and antibodies against NADPH-cytochrome P450 reductase. It was demonstrated that most ecdysone 20-monooxygenase activity was associated with microsomes, and that there was little or no intrinsic mitochondrial ecdysone 20-monooxygenase. Next, conditions for the measurement of ecdysone 20-monooxygenase activity were established for the microsomal fraction, and changes in the enzyme activity were measured in diapause eggs and non-diapause eggs during early embryogenesis. It was demonstrated that enzyme activity in diapause eggs remained at a low level, while that in the non-diapause eggs increased from the gastrula stage. The increase in egg ecdysone 20-monooxygenase activity was prevented by actinomycin D and alpha-amanitin, suggesting that gene transcription is required for eliciting an increase in ecdysone 20-monooxygenase activity.

Ecdysone 20-hydroxylation and 3-epimerization in larvae of the gypsy moth, Lymantria dispar L.: tissue distribution and developmental changes

The potential for ecdysone metabolism was determined for various larval tissues of the gypsy moth, Lymantria dispar. Homogenates of fat body, midguts, and Malpighian tubules, taken on different days during the second half of the fifth instar, were incubated with [ 3H]ecdysone, and the products were analyzed by reversed-phase and normal-phase HPLC. All tissues showed conversion to 20-hydroxyecdysone, and midguts also produced 3-epiecdysone. Ecdysone 20-monooxygenase (E20MO) activity in the fat body increased from a low level on day 5 to a peak on day 11, coinciding with the peak in the hemolymph ecdysteroid titer on the penultimate day of the instar. Midguts and Malpighian tubules showed E20MO activity only during the last 3 or 4 days of the instar, with the highest activity also occurring on the penultimate day. For the midguts, the appearance of the E20MO coincided with the transition from larval to pupal tissue. No activity was detected in larval midguts. 3-Epiecdysone formation, however, was mainly found in larval midguts, with only marginal activity detectable in pupal midguts.

Action of Different Ecdysteroids on the Regulation of mRNAs for the Ecdysone Receptor, MHR3, Dopa Decarboxylase, and a Larval Cuticle Protein in the Larval Epidermis of the Tobacco Hornworm, Manduca sexta

To determine which ecdysteroids may be biologically active in the larval epidermis of the tobacco hornworm, Manduca sexta,we studied the action of several known ecdysteroids and metabolites on the expression of the genes encoding the ecdysone receptor (EcR), Manducahormone receptor 3 (MHR3), dopa decarboxylase (DDC), and a larval cuticle protein (LCP-14). Both Day 2 fourth- and Day 2 fifth-instar larval epidermis contained significant 3β-reductase activity which metabolized 3-dehydroecdysone (3DE) and 3-dehydro-20-hydroxyecdysone (3D20E) to ecdysone (E) and 20-hydroxyecdysone (20E), respectively, but had only very low amounts of ecdysone oxidase activity (E to 3DE) and no detectable ecdysone 20-monooxygenase activity (E to 20E). When the expression of the various genes was studied in the epidermis in vitro,20E and 3D20E had similar effects, whereas E, 3DE, 26-hydroxyecdysone and 20,26-dihydroxyecdysone were ineffective. Exposure of Day 2 fifth-instar epidermis to 500 ng/ml of either 20E or 3D20E for 24 hr caused a rapid, biphasic increase in EcR-B1 mRNA. By contrast, EcR-A mRNA showed a less rapid initial increase followed by a slow steady rise and was less responsive to 3D20E. Ecdysone in a 1:1 mixture with 20E effectively halved the concentration of 20E needed to induce EcR-B1 mRNA but showed no synergism in the induction of EcR-A mRNA. The induction of MHR3 mRNA and of DDC mRNA in Day 2 fourth-instar epidermis as well as the suppression of DDC and LCP-14 gene expression by 3D20E was indistinguishable from that of 20E. Therefore, for Manducalarval epidermis, only 20E and 3D20E are biologically active ecdysteroids. Since the 3D20E can be converted to 20E by the epidermis, its effects are likely mediated by 20E.

Effects of the neem tree compounds azadirachtin, salannin, nimbin, and 6-desacetylnimbin on ecdysone 20-monooxygenase activity.

The effects of azadirachtin, salannin, nimbin, and 6-desacetylnimbin on ecdysone 20-monooxygenase (E-20-M) 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 fifth instar larvae of Manduca sexta were incubated with radiolabeled ecdysone and increasing concentrations (from 1 x 10(-8) to 1 x 10(-3) M) of the four compounds isolated from seed kernels of the neem tree, Azadirachta indica. All four neem tree compounds were found to inhibit, in a dose-dependent fashion, the E-20-M activity in three insect species. The concentration of these compounds required to elicit a 50% inhibition of this steroid hydroxylase activity in the three insect species examined ranged from approximately 2 x 10(-5) to 1 x 10(-3).

Ecdysone 20-hydroxylation inManduca sexta midgut: Kinetic parameters of mitochondrial and microsomal ecdysone 20-monooxygenases

The larval midgut of the tobacco hornworm, Manduca sexta, has high ecdysone 20-monooxygenase (E20MO) activity, located both in the mitochondria and in the microsomes. The apparent kinetic parameters for E20MO in mitochondria and microsomes were determined. The Km5 (for ecdysone) of the mitochondrial and microsomal enzymes were 1.63 × 10−5 and 3.67 × 10−7 M, respectively. The Vmax was 82.7 pmol/min/mg protein for mitochondria and 32.0 pmol/min/mg protein for microsomes. Although the mitochondrial E20MO has the higher Vmax, at physiological ecdysone concentrations (10−7 − 10−8 M) it is only one-eighth to one-tenth as active as the microsomal enzyme. It is concluded that the microsomal E20MO is the primary, if not the only, enzyme involved in ecdysone 20-hydroxylation in M. sexta midgut. © 1996 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Ecdysone 20-monooxygenase systems in flesh-flies (diptera: sarcophagidae), Neobellieria bullata and Parasarcophaga argyrostoma

1. 1. The ecdysone 20-monooxygenase activity is the highest in fat bodies of 0 hr third instar wandering stage flesh-fly larvae. 2. 2. After the white prepuparial stage, activity of ecdysone 20-monooxygenase systems decreases, almost to zero. 3. 3. Microsomal ecdysone 20-monooxygenase activity is maximal at pH 7.2 and 30°C, is dependent on an NADPH-regenerating system and is strongly inhibited by 0.01% Tween 80. 4. 4. Microsomal ecdysone 20-monooxygenase activity exhibits variation in activity during development, with peaks at birth, in the middle of the second instar, and at the beginning of wandering behaviour in the third instar.

Effects of plant flavonoids and other allelochemicals on insect cytochrome P-450 dependent steroid hydroxylase activity

The plant flavonoids flavone, chrysin, apigenin, kaempferol, morin, quercetin, myricetin and phloretin were found to inhibit in a dose-dependent manner the cytochrome P-450 dependent ecdysone 20-monooxygenase activity associated with adult female Aedes aegypti, wandering stage larvae of Drosophila melanogaster, and fat body and midgut from prewandering and wandering stage last instar larvae of Manduca sexta. The concentrations of these flavonoids required to elicit a 50% inhibition of the steroid hydroxylase activity in all the insects ranged from ca 1 × 10 −5 to 1 × 10 −3 M. In addition, lower concentrations (1 × 10 −6 to 1 × 10 −5 M) of the flavonols kaempfenol, morin, quercetin and myricetin significantly stimulated (50–100% above control) M. sexta fat body ecdysone 20-monooxygenase activity. Other plant allelochemicals examined and found to significantly inhibit insect ecdysone 20-monooxygenase activity include corynanthine, quinidine, and quinine; whereas, indican and mimosine were found to significantly stimulate M. sexta fat body steroid hydroxylase activity. Several allelochemicals were without effect at all concentrations tested. Although none of the compounds tested in this study elicited effects at very low concentrations (1 × 10 −9 to 1 × 10 −8 M), the in vitro monooxygenase radioassay does hold considerable promise as a screening tool for the detection and identification of plant allelochemicals which may function as biopesticides affecting insect ecdysteroidogenesis.

Early steps in ecdysteroid biosynthesis: Evidence for the involvement of cytochrome P-450 enzymes

The first step in the biosynthesis of ecdysteroids by Manduca sexta prothoracic glands, the conversion of cholesterol to 7-dehydrocholesterol, is mediated by an enzyme with characteristics of a microsomal cytochrome P-450, i.e. sensitivity to CO and fenarimol, and a requirement for NADPH. The enzyme responsible for hydroxylation at C-25 of the putative 3-dehydroecdysone precursor, 14-hydroxy-5β-cholest-7-en-3,6-dione, is also microsomal, while those mediating hydroxylations at C-22 and C-2 of 3,14,25-trihydroxy-5β-cholest-7-en-6-one are mitochondrial. Indirect evidence revealed that the steps between 7-dehydrocholesterol and the trideoxyecdysteroids occur in the mitochondria, suggesting that extensive shuttling of intermediates between the endoplasmic reticulum and mitochondria takes place in the prothoracic gland cell during ecdysteroid biosynthesis. During the fifth larval instar, cholesterol 7,8-dehydrogenase activity is evident from days 2 to 9, while the conversion to [ 3H]ecdysteroids is not significant prior to the ecdysteroid commitment peak on day 4. Terminal hydroxylase activity shows little change throughout the instar. These data support the hypothesis that regulation of the biosynthetic pathway by PTTH occurs at the step immediately following the formation of 7-dehydro-cholesterol. The steroid biosynthesis inhibitor, fenarimol, has been shown to inhibit each of these P-450 enzymes, as well as fat body ecdysone 20-monooxygenase, with an I 50 of 10 −4 M in disrupted glands, suggesting that it is a general P-450 inhibitor. The secretion of ecdysteroids by the glands in vitro is very sensitive to fenarimol, i.e. I 50 of 10 −6 M. RH5849, 1,2- dibenzoyl-1-tert-butylhydrazine, fails to inhibit any of these prothoracic gland reactions, yet strongly inhibits fat body ecdysone 20-monooxygenase activity. This suggests that RH5849 is a specific ecdysteroid substrate/product mimic in this reaction.

Effects of the adenylate cyclase activator forskolin and its inactive derivative 1,9-dideoxyforskolin on insect cytochrome P-450 dependent steroid hydroxylase activity

The adenylate cyclase activator forskolin and its pharmacologically inactive derivative 1,9-dideoxyforskolin were found to inhibit in a dose-dependent fashion the ecdysone 20-monooxygenase activity associated with wandering stage larvae of Drosophila melanogaster and fat body and midgut from last instar larvae of the tobacco hornworm, Manduca sexta. The concentrations of these labdane diterpenes required to elicit a 50% inhibition of the cytochrome P-450 dependent steroid hydroxylase activity in the insect tissues ranged from approximately 5 x 10(-6) to 5 x 10(-4) M.

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 hornworm, 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.