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

Horike, Nanao; Sonobe, Haruyuki

doi:10.1002/(sici)1520-6327(1999)41:1<9::aid-arch3>3.0.co;2-g

Cited by 37 publications

(19 citation statements)

References 26 publications

(33 reference statements)

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“…Furthermore, 20E was demonstrated to be produced both by de novo synthesis and dephosphorylation of maternal inactive ecdysteroid-phosphates (16 -18, 32). It was suggested that the hydroxylation at the C-20 position of E may be a late-limiting step of the de novo synthesis of 20E (17,32). Regarding the dephosphorylation of ecdysteroid-phosphates, there has been little information on the enzyme that specifically catalyzes dephosphorylation reaction not only in B. mori but also in other insect species, although lysosomal acid phosphatase prepared at the late stage of embryonic development has been reported to be capable of hydrolyzing E22P in S. gregaria (33,34).…”

Section: Discussionmentioning

confidence: 99%

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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: Discussionmentioning

confidence: 99%

“…Subcellular fractionation was performed by differential centrifugation according to previous studies (17,26) with some modifications. Three-day-old nondiapause eggs (1.0 g) were homogenized in 50 mM Tris-HCl buffer (2 ml), pH 7.5, containing 250 mM sucrose, 1 mM dithiothreitol, 0.5 mM PMSF, 10 M pepstatin A, and 10 M leupeptin.…”

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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“…Bombyx mori (Horike and Sonobe, 1999), Locusta migratoria (Tawfik et al, 2002), Chortoicetes terminfera (Gregg et al, 1987) and Oxya yezoensis (Kidokoro et al, 2006)]. However, the endocrine events associated with embryogenesis are species specific and there is considerable variation in ecdysteroid titres even between species from the same order (Whiting and Dinan, 1988).…”

Section: Endocrine and Signal Transductionmentioning

confidence: 97%

Embryonic diapause highlighted by differential expression of mRNAs for ecdysteroidogenesis, transcription and lipid sparing in the cricketAllonemobius socius

Reynolds

Hand

2009

Journal of Experimental Biology

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“…We previously reported that the content of 20-hydroxyecdysone in the diapause egg remains low, whereas that in the nondiapause egg increases significantly during development [4±6], and suggested that characterization of the ecdysone 20-hydroxylation reaction would be important in understanding silkworm embryogenesis [7]. Our subsequent studies have demonstrated that most of the 20-hydroxylation activity in the three-day-old nondiapause egg is associated with the microsomes [8], suggesting that the microsomal cytochrome P450-dependent hydroxylation system is involved in this metabolism.…”

mentioning

confidence: 70%

“…Ecdysone, 10 pmol, mixed with 0.2 mCi [ buffer, pH 7.5, containing 0.2 mm dithiothreitol and 0.2% BSA were added to the tube, which was kept on ice [8]. The tube was pre-incubated at 35 8C for 3 min, and then the reaction was initiated by the addition of 10 mL 1.5 mm NADPH.…”

Section: Assay For Ecdysone 20-hydroxylation Activitymentioning

confidence: 99%

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

Horike¹,

Takemori²,

Nonaka³

et al. 2000

Eur J Biochem

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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 fulllength cDNA obtained was composed of 3471 bp with an open reading frame encoding a protein of 687 aminoacid 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 NADPHdependent 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 NADPHcytochrome 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.

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

Cited by 37 publications

References 26 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

Embryonic diapause highlighted by differential expression of mRNAs for ecdysteroidogenesis, transcription and lipid sparing in the cricketAllonemobius socius

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

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