Isolation and identification of major ecdysteroid conjugates from the ovaries of Bombyx mori

Ohnishi, Eiji; Hiramoto, Masashi; Fujimoto, Yoshinori; Kakinuma, Katsumi; Ikekawa, Nobuo

doi:10.1016/0020-1790(89)90014-0

Cited by 30 publications

(15 citation statements)

References 13 publications

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“…Kinetic Analysis-Besides E22P, other ecdysteroid-phosphates, 20E22P, 2-deoxyecdysone 22-phosphate (2dE22P) and 22-deoxy-20-hydroxyecdysone 3-phosphate (22d20E3P), which were isolated from mature ovaries of B. mori (27,28), were also used as substrates for the kinetic study of EPPase. Substrate concentration was varied from 0.4 to 204.8 M. Amounts of the products 20E and 2-deoxyecdysone formed in the reaction mixture were determined by RIA using antiserum N-6 as described in the previous section; this antiserum scarcely cross-reacted to their phosphoric esters (Ͻ0.058% in 20E22P and Ͻ0.019% in 2dE22P).…”

Section: Methodsmentioning

confidence: 99%

Purification, Kinetic Characterization, and Molecular Cloning of a Novel Enzyme Ecdysteroid-phosphate Phosphatase

Yamada

Sonobe

2003

Journal of Biological Chemistry

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From eggs of the silkworm Bombyx mori, we isolated a novel enzyme that is involved in the conversion of physiologically inactive conjugated ecdysteroids, such as ecdysone 22-phosphate and 20-hydroxyecdysone 22-phosphate, to active free ecdysteroids. This enzyme, called ecdysteroid-phosphate phosphatase (EPPase), was located in the cytosol fraction and differed from nonspecific lysosomal acid phosphatases in various enzymic properties. EPPase was purified about 3,000-fold to homogeneity by seven steps of column chromatography. The cDNA clone encoding EPPase was isolated by reverse transcription polymerase chain reaction using degenerate primers on the basis of the partial amino acid sequence obtained from purified EPPase and by subsequent 3-and 5-rapid amplification of cDNA ends. The full-length cDNA of EPPase was found to be composed of 1620 bp with an open reading frame encoding a protein of 331 amino acid residues. A data base search showed that there was no functional protein with the amino acid sequence identical to that of EPPase. Northern blot analysis revealed that EPPase mRNA was expressed predominantly during gastrulation and organogenesis in nondiapause eggs but was not detected in diapause eggs whose development was arrested at the late gastrula stage. In nondiapause eggs, the developmental changes in the expression pattern of EPPase mRNA corresponded closely to changes in the enzyme activity and in the amounts of free ecdysteroids in eggs.In insects, ecdysteroids, which regulate development and reproduction, are synthesized in prothoracic glands. However, in many insect species, ecdysteroids have been demonstrated to be synthesized in ovaries as well, to accumulate in mature ovaries, and to be transferred to eggs (1-3). Most of these ecdysteroids are physiologically inactive phosphoric esters and synthesized by the enzyme, ATP:ecdysteroid-phosphotransferase (ecdysteroid kinase), which catalyzes phosphorylation of ecdysteroids ( Fig. 1) (4,5). It has been suggested that free ecdysteroids are liberated from maternal ecdysteroid-phosphates during embryonic development and participate in the secretion of serosal cuticle and/or the induction of embryonic cuticulogenesis (embryonic molt) in various insects, such as Locusta migratoria (6), Schistocerca greagaria (7), Blaberus craniifer (8), and Manduca sexta (9). Furthermore, the involvement of ecdysteroids in embryonic diapause has also been suggested in Lepidosphes ulmi (10), Bombyx mori (11), and L. migratoria (12).In B. mori, recently, it has been demonstrated that the continuous supply of 20-hydroxyecdysone (20E), 1 which has been demonstrated to be an active molecule in B. mori eggs (13), is required for embryonic development and that a deficiency of 20E induces embryonic diapause (13); that is, the cessation of embryonic development at the late gastrula stage (14,15). Furthermore, 20E was demonstrated to be formed by de novo synthesis (16 -18) and dephosphorylation of 20-hydroxyecdysone 22-phosphate (20E22P) and ecdysone 22-phosphate (E22P) (in the c...

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Section: Methodsmentioning

confidence: 99%

Purification, Kinetic Characterization, and Molecular Cloning of a Novel Enzyme Ecdysteroid-phosphate Phosphatase

Yamada

Sonobe

2003

Journal of Biological Chemistry

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“…It has been suggested that ecdysteroids phosphorylated at the C-22 position are storage forms from which active ecdysteroids can be released, whereas ecdysteroids phosphorylated at the C-2 position or the C-3 position are end products in ecdysteroid metabolism (18,19,29). In B. mori ovaries, only C-3 and C-22 phosphate esters have been detected (36,37,43,(57)(58)(59). We have already demonstrated that a crude preparation of B. mori ovaries has the capacity to phosphorylate the hydroxyl groups of the C-22 and C-3 positions using ecdysone, 2-deoxyecdysone, and 2,22-dideoxy-20-hydroxyecdysone as the phosphate acceptors: the former two ecdysteroids are phosphorylated at the C-22 position, and the latter is phosphorylated at the C-3 position (31).…”

Section: Discussionmentioning

confidence: 99%

“…Ecdysone 22-phosphate was synthesized chemically (34). Other ecdysteroids, 2-deoxyecdysone, 20-hydroxyecdysone, 22-deoxy-20-hydroxyecdysone, and 2,22-dideoxy-20-hydroxyecdysone, were extracted from B. mori ovaries and purified by high performance liquid chromatography (HPLC), as described previously (35)(36)(37).…”

Section: Methodsmentioning

confidence: 99%

“…In B. mori, ovaries have been found to contain four C-22 phosphate esters and three C-3 phosphate esters of ecdysteroids as follows: ecdysone 22-phosphate, 20-hydroxyecdysone 22-phosphate, 2-deoxyecdysone 22-phosphate, 2-deoxy-20-hydroxyecdysone 22-phosphate, 22-deoxy-20-hydroxyecdysone 3-phosphate, 2,22-dideoxy-20-hydroxyecdysone 3-phosphate, and a trace amount of 2,22-dideoxy-23-hydroxyecdysone 3-phosphate (29,36,37,43). Thus, using the free type of these ecdysteroid phosphates, substrate specificity of ecdysone 22-kinase could be analyzed.…”

Section: Biochemical Characterization Of Ecdysone 22-kinase-mentioning

confidence: 99%

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Purification, Kinetic Characterization, and Molecular Cloning of a Novel Enzyme, Ecdysteroid 22-Kinase

Sonobe

Ohira

Ieki

et al. 2006

Journal of Biological Chemistry

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This is the first report succeeding in the isolation and characterization of an enzyme and its gene involved in the phosphorylation of a steroid hormone. It has been demonstrated that ecdysteroid 22-phosphates in insect ovaries, which are physiologically inactive, serve as a "reservoir" that supplies active free ecdysteroids during early embryonic development and that their dephosphorylation is catalyzed by a specific enzyme, ecdysteroid-phosphate phosphatase (Yamada, R., and Sonobe, H. In steroid metabolism, it is generally accepted that cytochrome P450-mediated reaction, referred to as phase I metabolism, converts hydrophobic compounds into more polar metabolites, whereas phase II metabolism involves an adduct formation via a conjugation reaction. Phase II metabolism, such as sulfation, glucuronidation, and fatty acyl esterification, leads to a change of polarity or to a charge modification of the given metabolite, thereby modulating biological activity or facilitating elimination. In mammals, the sulfation pathway can be thought of as a reversible process, comprising two enzyme systems: the sulfotransferases, which catalyze the sulfation reaction, and the sulfatases, which catalyze the hydrolysis of sulfate esters formed by the action of sulfotransferases. Much is now known concerning the function of these enzyme systems as well as the molecular structure of the relevant cDNA and genes comprising these systems (1-4). However, to our knowledge, there exists very little information regarding the phosphorylation of steroid hormones in mammals.Ecdysteroids, which include the arthropod molting hormone, mediate a wide variety of developmental and reproductive events in insects (5). During the larval and pupal stages, ecdysteroids are synthesized in steroidogenic glands, known as the prothoracic glands (6). The detailed ecdysteroid biosynthetic pathway constituted by phase I metabolism is not fully clarified, but cytochrome P450 enzymes catalyzing the last four steps of the biosynthesis of 20-hydroxyecdysone (the principal molting hormone of arthropods) (i.e. CYP306a1 (25-hydroxylase), CYP302a1 (22-hydroxylase), CYP315a1 (2-hydroxylase), and CYP314a1 (20-hydroxylase)), have been identified and characterized in Drosophila melanogaster, Bombyx mori, and Aedes aeypti (7-13).Although glucosides (14), fatty acyl esters (15), and sulfate esters (16) of ecdysteroids are detected as minor products of phase II reaction in several insect species, the major products of the phase II reaction in most insect species are phosphate esters (17-20). The ovaries of most insect species have the capacity to accumulate ecdysteroid phosphates, which are physiologically inactive (21), in addition to the capacity to synthesize free ecdysteroids de novo (22,23). It has been suggested that the ecdystreoid phosphates that are accumulated in the ovaries are transferred to the eggs and function as a source of active free ecdysteroid before the prothoracic glands differentiate during embryonic development (24 -26). Recently, in B. mori eggs, a no...

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“…The purity of all of the purchased ecdysteroids was checked by high-performance liquid chromatography (HPLC, Sonobe et al, 1999;Makka and Sonobe, 2000). Other ecdysteroids, ecdysone 22-phosphate (E22P), 2-deoxyecdysone (2dE), 22-deoxy-20-hydroxyecdysone (22d20E), and 2-deoxy-20-hydroxyecdysone (2d20E), were extracted from B. mori eggs and purified by HPLC as described previously (Ohnishi et al, 1981(Ohnishi et al, , 1989Kamba et al, 1994).…”

Section: Ecdysteroidsmentioning

confidence: 99%

A possible role of 20‐hydroxyecdysone in embryonic development of the silkworm Bombyx mori

Makka

Seino

Tomita

et al. 2002

Arch Insect Biochem Physiol

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It has been well established that eggs of insects, including those of the silkworm Bombyx mori, contain various ecdysteroids and the amounts of these ecdysteroids fluctuate during embryonic development. In order to know the function of egg ecdysteroids in embryonic development of B. mori, we examined the biological activities of various egg ecdysteroids by in vitro ligand-binding assay and bioassay using B. mori eggs. First, using the ecdysteroid receptor of B. mori (BmEcR-B1/BmUSP heterodimer) prepared by yeast and Escherichia coli expression systems, the interaction between the ecdysteroid receptor and various egg ecdysteroids of B. mori was analyzed. The relative binding affinities of egg ecdysteroids to the BmEcR-B1/BmUSP heterodimer decreased in the order of 20-hydroxyecdysone > 2-deoxy-20-hydroxyecdysone > 22-deoxy-20-hydroxyecdysone > ecdysone > 2-deoxyecdysone > ecdysone 22-phosphate. Next, several egg ecdysteroids of B. mori were injected into the prospective diapause eggs, which show a very low level of free ecdysteroids at the onset of embryonic diapause (gastrula stage). Approximately 7% of them (P < 0.002, chi(2)-test) developed beyond the gastrula stage without entering diapause by the injection of 20-hydroxyecdysone (25 ng/egg). In contrast, the injection of other ecdysteroids was not effective in inducing embryonic development. These results suggest that 20-hydroxyecdysone, via the ecdysteroid receptor, is responsible for the developmental difference between diapause and non-diapause in B. mori embryos. Furthermore, it was suggested that continuous supply of 20-hydroxyecdysone may be required to induce embryonic development.

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Isolation and identification of major ecdysteroid conjugates from the ovaries of Bombyx mori

Cited by 30 publications

References 13 publications

Purification, Kinetic Characterization, and Molecular Cloning of a Novel Enzyme Ecdysteroid-phosphate Phosphatase

Purification, Kinetic Characterization, and Molecular Cloning of a Novel Enzyme Ecdysteroid-phosphate Phosphatase

Purification, Kinetic Characterization, and Molecular Cloning of a Novel Enzyme, Ecdysteroid 22-Kinase

A possible role of 20‐hydroxyecdysone in embryonic development of the silkworm Bombyx mori

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