In vitro secretion of ecdysteroids by Y-organs of the crayfish,Procambarus clarkii

Sonobe, Haruyuki; Kamba, Mari; Ohta, Keisuke; Ikeda, Mineo; Naya, Yoko

doi:10.1007/bf01929889

Cited by 44 publications

(26 citation statements)

References 12 publications

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“…Therefore, the amount of E in the reaction mixture was quantified by RIA without isolating E from E22P. RIA was conducted as described previously (20). For the measurement of EPPase activity, the reaction mixture was made up to a final volume of 100 l with 200 mM Tris-HCl buffer, pH 7.5, containing 0.1% bovine serum albumin, 100 nM E22P, and 50 l of the enzyme solution.…”

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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“…To enhance the sensitivity to radioimmunoassay using the antiserum N6 (40), ecdysteroid phosphates were hydrolyzed to free ecdysteroids, using a digestive enzyme preparation from Helix pomatia (see below). The radioimmunoassay was conducted as described previously (41). Ecdysteroid quantity was expressed in terms of ecdysone equivalents.…”

Section: Methodsmentioning

confidence: 99%

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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“…Molt-inhibiting activity was measured using an in vitro culture of Y-organs of the crayfish, Procambarus clarkii, and an ecdysteroid radioimmunoassay (RIA) (Sonobe et al 1991). In this work, a pair of Y-organs was dissected out of an eyestalk-ablated animal.…”

Section: -2a Bioassay and Purificationmentioning

confidence: 99%

Crustacean Peptide Hormones: Structure, Gene Expression and Function

Katayama¹,

Ohira²,

Nagasawa³

2013

Aqua-BioSci. Monogr. (ABSM)

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Keywords AbstractHomeostasis, growth and reproduction in crustaceans are under endocrine control as is established in other animals. The identification of crustacean hormones and elucidation of mechanisms of hormonal regulation have been pursued extensively. Mechanisms are found to be partly similar to, but also considerably different from those in insects, although both groups of animals are arthropods. Molting in insects, for example, is positively regulated by prothoracicotropic hormone (PTTH), which is secreted by the brain/ corpora allata complex and stimulates the prothoracic glands to promote the synthesis and secretion of ecdysteroids. On the other hand, molting in crustaceans is negatively regulated by molt-inhibiting hormone (MIH), which is secreted by the X-organ/sinus gland complex and suppresses the Y-organs from synthesizing ecdysteroids during the intermolt period. Therefore, it is rather difficult to fully elucidate physiological mechanisms in crustaceans based only on the results obtained in insects. Using experimental evidence, we have thus far looked into the roles of crustacean eyestalk hormones and androgenic gland hormone (AGH). Various eyestalk hormones have been isolated from the sinus glands and characterized: e.g., crustacean hyperglycemic hormone (CHH), MIH, vitellogenesis-inhibiting hormone (VIH), red pigment concentrating hormone (RPCH) and pigment dispersing hormone (PDH). CHH, MIH and VIH are structurally similar, forming the CHH-family. Insects also possess molecules similar to CHH, RPCH and PDH, but their functions are different. In addition, AGH has been studied mainly using terrestrial isopods. Here, we describe the present state of our understanding of the structure and function of these peptide hormones, and discuss future perspectives. mercial importance in fisheries and aquaculture, while others play a key role as intermediates in the food chain, linking phytoplanktons and fishes within the aquatic ecosystem. Modern shrimp culture was first established in Japan over 50 years ago using the kuruma prawn Marsupenaeus (formerly Penaeus) japonicus, which is one of the most important commercial species in Japan, and the aquaculture technique has now spread to many countries, especially in tropical and subtropical

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In vitro secretion of ecdysteroids by Y-organs of the crayfish,Procambarus clarkii

Cited by 44 publications

References 12 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

Crustacean Peptide Hormones: Structure, Gene Expression and Function

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