Evidence for an α-ecdysone cytochrome P-450 mixed function oxidase in insect fat body mitochondria

Bollenbacher, Walter E.; Smith, Stan L.; Wielgus, John J.; Gilbert, Lawrence I.

doi:10.1038/268660a0

Cited by 94 publications

(24 citation statements)

References 14 publications

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“…For Dib and Sad, this is consistent with prior biochemical fractionation data showing that both the 22-and 2-monooxygenase activities are mitochondrial (29). Prior cell fractionation data for the E20MO is more ambiguous, with a microsomal and͞or mitochondrial localization identified, depending on the insect, tissue, and developmental stage being examined (4,5,7,46,47). The N terminus of the Shd protein contains both hydrophobic signal-type sequences typical of microsomal P450s (48) as well as a charged segment containing sequences characteristic of typical mitochondrial enzymes (49).…”

Section: Discussionsupporting

confidence: 75%

“…3 and Tables 2 and 3 also indicate that Shd is responsible for all E20MO activity during larval and adult stages, because it is expressed in tissues (fat body, midgut, Malpighian tubules, epidermis, salivary glands, and ovaries) known to possess this enzyme activity in vitro during these stages, not only in Drosophila (30)(31)(32)(33)(34) but also in other Dipteran insects (35). In addition, shd is not expressed in tissues where such activity has been reported to be absent, such as in the ring glands, brain, ventral ganglion, and muscle of Diptera and other insects including Drosophila (5,(36)(37)(38)(39)(40)(41)(42). The complete absence of in vitro E20MO activity in the larval fat body, Malpighian tubules, salivary glands, and adult ovaries of rescued homozygous UAS-shd͞twistGal4 flies relative to that observed in corresponding heterozygous UAS-shd͞twistGal4, yw, or wild-type tissues is presented as proof that Shd is solely responsible for E20MO activity in these tissues.…”

Section: Discussionmentioning

confidence: 99%

“…Although it has been known for several decades that the E20MO is a P450 enzyme that is localized in the endoplasmic reticulum (4) and͞or mitochondria (5,6), depending on the insect, tissue, and developmental stage (7,8), it has not been purified to homogeneity nor has the gene coding for this enzyme been cloned. Here, we report the cloning of shade (CYP314a1), a member of the Halloween gene family (9), and demonstrate that its gene product codes for the Drosophila E20MO.…”

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confidence: 99%

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Shade is the Drosophila P450 enzyme that mediates the hydroxylation of ecdysone to the steroid insect molting hormone 20-hydroxyecdysone

Petryk

Warren

Marqués

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

413

373

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The steroid 20-hydroxyecdysone (20E) is the primary regulatory hormone that mediates developmental transitions in insects and other arthropods. 20E is produced from ecdysone (E) by the action of a P450 monooxygenase that hydroxylates E at carbon 20. The gene coding for this key enzyme of ecdysteroidogenesis has not been identified definitively in any insect. We show here that the Drosophila E-20-monooxygenase (E20MO) is the product of the shade (shd) locus (cytochrome p450, CYP314a1). When shd is transfected into Drosophila S2 cells, extensive conversion of E to 20E is observed, whereas in sorted homozygous shd embryos, no E20MO activity is apparent either in vivo or in vitro. Mutations in shd lead to severe disruptions in late embryonic morphogenesis and exhibit phenotypes identical to those seen in disembodied (dib) and shadow (sad) mutants, two other genes of the Halloween class that code for P450 enzymes that catalyze the final two steps in the synthesis of E from 2,22-dideoxyecdysone. Unlike dib and sad, shd is not expressed in the ring gland but is expressed in peripheral tissues such as the epidermis, midgut, Malpighian tubules, and fat body, i.e., tissues known to be major sites of E20MO activity in a variety of insects. However, the tissue in which shd is expressed does not appear to be important for developmental function because misexpression of shd in the embryonic mesoderm instead of the epidermis, the normal embryonic tissue in which shd is expressed, rescues embryonic lethality.

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Section: Discussionsupporting

confidence: 75%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Shade is the Drosophila P450 enzyme that mediates the hydroxylation of ecdysone to the steroid insect molting hormone 20-hydroxyecdysone

Petryk

Warren

Marqués

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

413

373

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“…The fat body enzyme is located in the mitochondria (Bollenbacher et al, 1977;Smith et al, 1979Smith et al, ,1980. The midgut contains two different E20MOs, one in the mitochondria and one in the microsomes.…”

Section: Introductionmentioning

confidence: 99%

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

Weirich

Williams

Feldlaufer

1996

Arch. Insect Biochem. Physiol.

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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.

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“…Ecdysone 20-monooxygenase activity has been characterized in vitro in various insect tissues. The highest activities were recorded in the fat body [4,5], the gut [6-81 and the Malpighian tubules [3,9,10]. No conversion of ecdysone into 20-hydroxyecdysone was detected in imaginal discs [ll-141.…”

Section: Introductionmentioning

confidence: 99%

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

Blais¹,

Lafont²

1986

Arch Insect Biochem Physiol

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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. I t s apparent Km i s 58 n M for ecdysone. The enzyme is inhibited by the reaction product 20-hydroxyecdysone with an apparent Ki of 2.6 pM. 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 i s 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.

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Evidence for an α-ecdysone cytochrome P-450 mixed function oxidase in insect fat body mitochondria

Cited by 94 publications

References 14 publications

Shade is the Drosophila P450 enzyme that mediates the hydroxylation of ecdysone to the steroid insect molting hormone 20-hydroxyecdysone

Shade is the Drosophila P450 enzyme that mediates the hydroxylation of ecdysone to the steroid insect molting hormone 20-hydroxyecdysone

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

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

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