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.     

Ecdysone 20-hydroxylation in imaginal wing discs of Pieris brassicae (lepidoptera): Correlations with ecdysone and 20-hydroxyecdysone titers in pupae

Ecdysone 20-hydroxylase activity has been detected in pupal wing discs of Pieris brassicae. This activity is due to an enzyme system located in microsomal fractions. Its apparent Km is 58 nM for ecdysone. The enzyme is inhibited by the reaction product 20-hydroxyecdysone with an apparent Ki of 2.6 μM. Its activity varied during pupal-adult development with a maximum on day 4, when ecdysone levels are the highest in the animal. Although low, the peak activity is sufficient to assure 25% of the conversion of endogenous ecdysone into 20-hydroxyecdysone in pupae. Ecdysone and 20-hydroxyecdysone levels were measured in hemolymph and whole animals; ecdysone appears to be mainly located in hemolymph, whereas 20-hydroxyecdysone seems to be equally distributed between hemolymph and tissues. All these findings are discussed in relation to the roles of ecdysone and 20-hydroxyecdysone during pupal-adult development.     

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.     

Comparative studies on ecdysone metabolism between mature larvae and pharate pupae in the fleshfly,Sarcophaga peregrina

Metabolites of radioactive ecdysone or 20-hydroxyecdysone in larvae and pharate pupae of Sarcophaga peregrina were separated and identified by using thin-layer chromatography, high-performance liquid chromatography, and chemical methods. At the larval stage ecdysone was metabolized to biologically less active ecdysteroids predominantly through 20-hydroxyecydsone, at the pharate pupal stage, to other ecdysteroids which were tentatively identified as 26-hydroxyecdysone, 3-epi-26-hydroxyecdysone, and 3-epi-20,26-dihydroxyecdysone. Ecdysteroid acids were found in the polar metabolites during pharate pupal-pupal transformation, but scarcely detected in the larval metabolites. These acids were presumed to be ecdysonoic acid, 20-hydroxyecdysonoic acid, and their epimers. The conjugates of ecdysteroid that released the free ecdysteroids by enzymatic hydrolysis were produced more in larvae than in pupae, whereas the very polar ecdysteroids that were not affected by the enzyme were found more in pupae. Therefore, there are different metabolic pathways of ecdysone between these two successive developmental stages, and the alteration of the metabolic pathway may serve as one of the important factors in a regulatory mechanism of molting hormone activity which is responsible for normal development of this insect.     

Identification of the 22-phosphate esters of ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone from newly laid eggs of the desert locust, Schistocerca gregaria.

The four major ecdysteroid (insect moulting hormone) conjugates present in the newly laid eggs of the desert locust, Schistocera gregaria, have been purified by reversed-phase and anion-exchange high-performance liquid chromatography. The steroid moieties were identified as ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone. Phosphate analysis of acid-hydrolysed samples showed a steroid:phosphate ratio of approx. 1:1 for all four compounds. The intact conjugates were identified as ecdysone 22-phosphate, 2-deoxyecdysone 22-phosphate, 20-hydroxyecdysone 22-phosphate and 2-deoxy-20-hydroxyecdysone 22-phosphate by fast atom bombardment mass spectrometry and 1H, 13C and 31P n.m.r. The significance of ecdysteroid phosphates as a source of free hormone during embryogenesis is discussed.     

Feedback inhibition of ecdysone production by 20-hydroxyecdysone in Pieris brassicae pupae

The effects of exogenous moulting hormones, ecdysone and 20-hydroxyecdysone on ecdysteroid production were studied in vivo in Pieris brassicae pupae. Both hormones inhibit ecdysteroid production; however, 20-hydroxyecdysone is much more efficient than ecdysone, and it is likely that the ecdysone effect is due to its partial conversion into 20-hydroxyecdysone. These results suggest that 20-hydroxyecdysone acts on ecdysteroid production as a negative-feedback regulator. Furthermore, since 20-hydroxyecdysone elicits inhibition in headless pupae, it is suggested that 20-hydroxyecdysone acts directly upon the prothoracic glands.     

The ecdysteroids from young embryonated eggs of the tobacco hornworm

26-Hydroxyecdysone, which is the major free recoverable ecdysteroid of older age groups of embryonated eggs of the tobacco hornworm was also the major component in 4- to 18-hour-old embryonated eggs. The other 3β-ecdysteroids, ecdysone, 20-hydroxyecdysone, and 20,26-dihydroxy-ecdysone, were also present and accounted for an the molting hormone activity; 26-hydroxyecdysone was devoid of molting hormone activity in the house fly assay. 20-Hydroxyecdysone was a minor component, which confirms the earlier observations that the main metabolic route for ecdysteroids during embryonic development is that leading to 26-hydroxy-ecdysone, whereas formation of 20-hydroxyecdysone is a minor pathway. A new 3α-ecdysteroid, 3-epi-26-hydroxyecdysone, also devoid of molting hormone activity, was the second major ecdysteroid isolated from the eggs. 3-Epi-20,26-dihydroxyecdysone was detected in very minute amounts. In additon to the six 3β-and 3α-ecdysteroids there were at least an equivalent number of unknown ecdysteroids an of which lacked molting hormone activity. Their physical properties including chromatographic behavior are discussed.     

The identification and titres of conjugated and free ecdysteroids in developing ovaries and newly-laid eggs of Schistocerca gregaria

Ecdysteroid levels throughout ovarian development and in newly-laid eggs of S. gregaria have been determined. A simple method for the separation of free and conjugated ecdysteroids is described. Both free and polar conjugated ecdysteroids are present at the end of oögenesis and in newly-laid eggs, but the polar conjugated ecdysteroids always predominate; 95% of the total ecdysteroid in newly-laid eggs is in the conjugated form. Ecdysone, 2-deoxyecdysone and 20-hydroxyecdysone have been fully characterized from both the ‘free’ and ‘conjugated’ fractions. The presence of traces of 26-hydroxyecdysone in the ‘conjugate’ fraction was indicated by HPLC analyses. The levels of ecdysteroid released from the conjugates of newly-laid eggs were 35 μg/egg pod (44 μg/g wet weight) for ecdysone, 16 μg/egg pod (19.4 μg/g) for 2-deoxyecdysone and 5 μg/egg pod (6.1 μg/g) for 20-hydroxyecdysone. The level of free ecdysone found in newly-laid eggs was 2 μg/egg pod (2.6 μg/g).     

Expression of the Csp protein family upon cold shock and production of tetracycline in Streptomyces aureofaciens

The biosynthesis of the steroidal molting hormone, 20-hydroxyecdysone, of arthropods involves a series of cytochrome P450-catalyzed hydroxylations. None of the many sequences of insect cytochromes P450, known to date, is related to ecdysteroid pathways. Here, we report the cloning and sequencing of a full-length cDNA of a new cytochrome P450, classified as CYP6H1, from malpighian tubules of the locust, Locusta migratoria. The 1854 bp DNA contained an open reading frame coding for a protein of 542 amino acids, a 5'-leader sequence and a 3'-untranslated region containing a polyadenylation signal and a poly(A) tail. The encoded protein had been isolated as an ecdysone-binding cytochrome P450 from microsomes of the same tissue in previous work. The closest homolog of CYP6H1 was CYP6A2 from Drosophila with 42.1% identity. According to Northern analysis, CYP6H1 is predominantly expressed at larval instars and in malpighian tubules. Evidence is presented for a functional assignment of CYP6H1 to microsomal ecdysone 20-hydroxylase of the locust.     

Characterization of acetyl-CoA: Ecdysone 3-acetyltransferase in Schistocerca gregaria larvae

Characterization of the acetyltransferase (acetyl-CoA: ecdysone 3-acetyltransferase) which catalyzes the conversion of ecdysone into ecdysone 3-acetate was carried out in gastric caecae of day 7 last instar larvae of Schistocerca gregaria. This enzyme is one of the enzymic systems involved in the inactivation of ecdysteroids. The acetyltransferase exhibited a microsomal subcellular localization, an apparent K_m for ecdysone of 71 μM, a maximal specific activity of 7.2 nmol/min/mg of protein and was inhibited competitively in the presence of 20-hydroxyecdysone with K_i = 68.8 μM. The enzyme required acetyl-CoA as co-substrate for its activity, the apparent K_m for acetyl-CoA being 47.2 μM. Acetic acid could not replace acetyl-CoA as the co-substrate, indicating that the enzyme is an acetyl-CoA: ecdysone acetyltransferase and not a hydrolase. Similarly, esterification of ecdysone was not observed when long-chain fatty acyl-CoA derivatives were substituted as co-substrates. The reaction was linear for 20 min and with protein concentration up to 0.8 mg/ml.The formation of 20-hydroxyecdysone 3-acetate has been demonstrated in the same microsomal fraction and required also acetyl-CoA as co-substrate. The apparent K_m of the acetyltransferase for 20-hydroxyecdysone was 53.5 μM, revealing that the enzyme had a somewhat stronger affinity for 20-hydroxyecdysone than for ecdysone.     

Metabolism of [3H]-ecdysone in Schistocerca gregaria; formation of ecdysteroid acids together with free and phosphorylated ecdysteroid acetates

The metabolism of [³H]-ecdysone has been investigated at times of low and high endogenous ecdysteroid tit re, in early and late fifth-instar Schistocerca gregaria larvae, respectively. Ecdysone-3-acetate, 20-hydroxyecdysone, and 20,26-dihydroxyecdysone were identified as metabolites in both the free form and as polar conjugates. Comparison of the intact polar conjugates of the ecdysteroid acetates on two HPLC systems with the corresponding authentic compounds indicated that they were 3-acetylecdysone-2-phosphate and 3-acetyl-20-hydroxyecdysone-2-phosphate. Other major polar metabolites were identified as ecdysonoic acid and 20-hydroxyecdysonoic acid. Ecdysone metabolism in fifth-instar S. gregaria is apparently an age-dependent process. Early in the instar, excretion of both free and conjugated ecdysteroids, as well as ecdysteroid 26-acids, occurs. At this stage the level of ecdysteroid acetates in the conjugated (phosphate) form is high, in contrast to the free ecdysteroids, where ecdysone predominates. When the endogenous hormone titre is high, the formation of ecdysteroid acetates is less, the major excreted matabolites at that stage being conjugated 20-hydroxyecdysone together with ecdysteroid-26-acids, but little free ecdysteroids. Acetylation of ecdysone occurs primarily in the gastric caecae. Ecdysone-3-acetate (mainly as polar conjugate) is also a major product of ingested ecdysone in early fifth-instar Locusta migratoria.     

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.     

Metabolism of ecdysteroid by testes of the tobacco budworm,Heliothis virescens

Testes from late last stage larvae of the tobacco budworm, Heliothis virescens, were incubated with [³H]ecdysone and [³H]cholesterol. [³H]Ecdysone was converted to six other major ecdysteroids, identified by cochromatography in reverse-phase high-pressure liquid chromatography (RPHPLC); four of them were verified by normal-phase HPLC. A highly polar fraction, moderately polar ecdysteroids (20,26-dihydroxyecdysone, 3-epi-20-hydroxyecdysone, and 20-hydroxyecdysone) and low-polarity ecdysteroids, including 2-deoxyecdysone, were detected after incubation with [³H]ecdysone. Compounds that reacted positively to antibodies to progesterone and testosterone were detected in the low-polarity fractions. Testes were incubated in fractions corresponding to each of the major ecdysteroid peaks derived from [³H]ecdysone metabolism. Although most of the radioactive ecdysteroid fractions were further metabolized to high- and low-polarity endpoints, 88% of the [³H]20-hydroxyecdysone peak apparently remained unmetabolized. 20-Hydroxyecdysone may be the primary ecdysteroid product of testes of H. virescens. [³H]Cholesterol was not metabolized to any appreciable extent.     

Ecdysone metabolism in Pieris brassicae during the feeding last larval instar

Ecdysone metabolism in Pieris brassicae during the feeding last larval stage was investigated by using ³H-labeled ecdysteroid injections followed by high-performance liquid chromatographic (HPLC

1 Abbreviations: 3DE = 3-dehydroecdysone; 3D20E = 3-dehydro-20-hydroxyecdysone; 2026E = 20,26-dihydroxyecdysone; E = ecdysone; Eoic = ecdysonoic acid; 2026E′ = 3-epi-20,26-dihydroxyecdysone; E′ = 3-epiecdysone; E′oic = 3-epiecdysonoic acid; E′8P = 3-epiecdysone 3-phosphate; 20E′ = 3-epi-20-hydroxyecdysone; 20E′3P = 3-epi-20-hydroxyecdysone 3-phosphate; FT = Fourier transform; HPLC = high-performance liquid chromatography; 20E = 20-hydroxyecdysone; 20Eoic = 20-hydroxyecdysonoic acid; NMR = nuclear magnetic resonance; NP-HPLC = normal phase HPLC; RP-HPLC = reverse phase HPLC; TFA = trifluoroacetic acid; Tris = tris(hydroxymethyl)-aminomethane.

) analysis of metabolites. Metabolites were generally identified by comigration with available references in different HPLC systems. Analysis of compounds for which no reference was available required a large-scale preparation and purification for their identification by ¹H nuclear magnetic resonance spectrometry. The metabolic reactions affect the ecdysone molecule at C-3, C-20, and C-26, leading to molecules which are modified at one, two, or three of these positions. At C-20, hydroxylation leads to 20-hydroxyecdysteroids. At C-26, hydroxylation leads to 26-hydroxyecdysteroids which can be further converted into 26-oic derivatives (ecdysonoic acids) by oxidation. At C-3, there are several possibilities: there may be oxidation into 3-dehydroecdysteroids, or epimerization possibly followed by phosphate conjugation. Thus, injected 20-hydroxyecdysone was converted principally into 20-hydroxyecdysonoic acid, 3-dehydro-20-hydroxyecdysone, and 3-epi-20-hydroxyecdysone 3-phosphate. Labelled ecdysone mainly gave the same metabolites doubled by a homologous series lacking the 20-hydroxyl group.     

Ecdysone metabolism and distribution during the pupal-adult development of Manduca sexta

The metabolism and distribution of endogenous ecdysone and injected [³H]ecdysone were studied during the pupal-adult development of Manduca sexta. Well-characterized antisera were used to detect and quantify endogenous metabolites by radioimmunoassay (RIA) following their separation by ion-suppressed reverse phase, and normal phase, high performance liquid chromatography. Identical chromatographic procedures were employed to determine the metabolic fate of the [³H]ecdysone in the haemolymph pool. These studies revealed the sequential appearance in the haemolymph and gut of progressively oxidized metabolites of ecdysone—hydroxylation at C-20 was followed by hydroxylation at C-26. The data are suggestive of both the induction of the steroid hydroxylases (oxidases) by substrate or other effector substances and the possible coordination of developmental events by ecdysteroids other than 20-hydroxyecdysone.In the haemolymph, two highly-polar conjugates of ecdysone were observed together with conjugates of the other free ecdysteroids, especially those hydroxylated at C-26. In contrast, relatively little 20-hydroxycdysone conjugate was detected in the insect. As adult development proceeded, both endogenous and radiolabelled ecdysteroids were increasingly localized in the gut, so that just prior to eclosion most ecdysteroids were present in the meconium of the high gut (rectal pouch). The peak titres and the kinetics of appearance of ecdysone, 20-hydroxyecdysone, and 20,26-dihydroxyecdysone were similar for both haemolymph and gut (and for males and females), but considerably higher levels of C-26 oxidized (acid) metabolites of ecdysone and 20-hydroxyecdysone were localized in the gut. Although levels of highly-polar ecdysteroid conjugates found in the haemolymph and gut were similar, considerable amounts of three less polar ecdysone conjugates, of 3-α-epimers of ecdysone and 20-hydroxyecdysone, and of a substance tentatively identified as 2-deoxyecdysone were found only in the gut. Whether ionized, conjugated, or free, the gut ecdysteroids did not appear to equilibrate with the haemolymph compartment.Differences were observed in the metabolism kinetics of exogenously administered radiolabelled ecdysone when compared to the endogenous ecdysteroids; and some RIA positive gut metabolites did not become significantly radiolabelled. This suggests that injection of ecdysone may not simulate the endogenous secretion of ecdysone or its subsequent metabolism and distribution completely accurately.     

Location of ecdysteroid 22-O-acyltransferase in the larvae ofHeliothis virescens

Larvae of the tobacco budworm,Heliothis virescens, are resistant to high levels of ingested 20-hydroxyecdysone which could cause potential inhibition to the development of many other lepidopteran species. This resistance is attributed to the ability of the larvae to metabolize this molting hormone to its 22-acyl ester forms. When tobacco budworm larvae were fed large quantities of 20-hydroxyecdyone, the hormonal metabolites were found in gut and fat body tissues. When incubated with 20-hydroxyecdysone gut tissue converted 20-hydroxyecdysone into its 22-acyl ester metabolites. Lumen site of the midgut was found to be the major location of this bio-transformation. In contrast, fat body tissue failed to convert 20-hydroxyecdysone to 22-acyl ester metabolitesin vitro. After the oral injection of³H-ecdysone, the major metabolites formed were ecdysone 22-acyl esters whereas the majority of³H-ecdysone was transformed to polar metabolites after it was injected into the hemocoel of the larvae. Similar distributions of ecdysteroid 22-O-acyltransferase and alkaline phosphatase activity in subcellular fractions demonstrates the co-localization of these enzymes in plasma membrane of the gut epithelial cells. These results suggest that gut brush border membrane is the major site of ecdysteroid 22-acyl ester formation inH. virescens larvae.     

Meeting announcements

The levels of individual free and conjugated ecdysteroids and ecdysteroid acids, labeled from [¹⁴C]cholesterol, in five different age groups of male Manduca sexta during pupal-adult development were determined by HPLC. Eight free ecdysteroids, eight ecdysteroid phosphates, and two ecdysteroid acids were identified. Newly ecdysed pupae contained predominantly 3-epiecdysteroids in each of the free, conjugated, and acidic ecdysteroid fractions. The titer of each ecdysteroid fraction rose sharply by day 4, and this was particularly noteworthy with respect to free ecdysone and 3-epi-20-hydroxyecdysonoic acid. This stage demonstrated high degrees of ecdysone biosynthesis, oxidative catabolism, and phosphorylation. As development proceeded to day 16, total ecdysteroid titer remained constant; a decreasing free ecdysteroid titer was accompanieid by increasing titers of both conjugates and acids resulting from the metabolic processes of hydroxylation, oxidation, epimerization, and phosphorylation. The predominant metabolites throughout development were 3-epi-20-hydroxyecdysonoic acid and the phosphate conjugates of 3-epi-20-hydroxyecdysone and 3-epi-20,26-dihydroxyecdysone. The ultimate inactivation of the ecdysteroids of M. sexta during pupal-adult development is possibly mediated by two pairs of metabolically-linked processes, one leading to a 3-epiecdysteroid acid, and the other to 3-epiecdysteroid phosphates.