Ecdysone 3-epimerase from the midgut of Manduca sexta (L.)

Mayer, Richard T.; Durrant, J L; Holman, G. Mark; Weirich, Günter F.; Svoboda, James A.

doi:10.1016/s0039-128x(79)80016-1

Cited by 31 publications

(27 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Also present was a shorter product that corresponded approximately in length to the largest product of the cytoplasmic expression system ( Figure 4B). Kinetic parameters (Table 4) for cytochrome c reductase activity of the purified protein were similar to those previously reported for house fly reductase purified from microsomes (Mayer & Durrant, 1979;Vincent & Terriere, 1985).…”

Section: B (Bottom): Difference Spectrum Of the Ferrous Co Complex Ofsupporting

confidence: 82%

Expression of Housefly CYP6A1 and NADPH-Cytochrome P450 Reductase in Escherichia coli and Reconstitution of an Insecticide-Metabolizing P450 System

Andersen

Utermohlen²,

Feyereisen³

1994

Biochemistry

120

View full text Add to dashboard Cite

The house fly (Musca domestica) cytochrome P450 gene CYP6A1 was expressed in Escherichia coli. The native protein was produced at a level of 0.25-0.34 mumol/L (15-20 mg/L) of culture with approximately 50% of the P450 being associated with the membrane fraction. The CYP6A1 protein was characterized spectrally and purified by a combination of hydrophobic interaction and hydroxyapatite chromatography. The house fly NADPH-cytochrome P450 reductase gene was also expressed in E. coli. Expression of a cytoplasmically directed reductase resulted in a protein that reduced cytochrome c but did not support P450 monooxygenase reactions. However, a periplasmically directed reductase was found to support monooxygenase reactions with CYP6A1 in a reconstituted system. The reconstituted system was effective in the epoxidation of the cyclodiene insecticides aldrin and heptachlor, with turnover rates of 12 and 34 min-1, respectively. The enzyme showed little detectable activity in the O-dealkylation and N-dealkylation of various compounds that are metabolized by house fly microsomes. Incubation with polyclonal antisera raised against purified CYP6A1 inhibited the microsomal epoxidation of heptachlor by 65%. Under the same conditions, the metabolism of 7-methoxy-4-methylcoumarin was inhibited only slightly. The results suggest that CYP6A1 is a major cyclodiene epoxidase in the house fly and that multiple P450 forms are responsible for the elevated monooxygenase activities in insecticide-resistant flies.

show abstract

Section: B (Bottom): Difference Spectrum Of the Ferrous Co Complex Ofsupporting

confidence: 82%

Expression of Housefly CYP6A1 and NADPH-Cytochrome P450 Reductase in Escherichia coli and Reconstitution of an Insecticide-Metabolizing P450 System

Andersen

Utermohlen²,

Feyereisen³

1994

Biochemistry

120

View full text Add to dashboard Cite

show abstract

“…The low stimulatory effect produced by increasing salt concentrations is not characteristic of the artichoke reductase, since it was also observed for yeast (Kubota et al, 1977), housefly (Wilson & Hodgson, 1971;Mayer & Prough, 1977;Mayer & Durrant, 1979), southernarmyworm midgut (Crankshaw et al, 1979) and guinea-pig liver (Kobayashi & Rikans, 1984) reductases.…”

Section: Discussionmentioning

confidence: 85%

Purification and characterization of the NADPH-cytochrome P-450 (cytochrome c) reductase from higher-plant microsomal fraction

Benveniste¹,

Gabriac²,

Durst³

1986

Biochemical Journal

View full text Add to dashboard Cite

NADPH-cytochrome P-450 (cytochrome c) reductase (EC 1.6.2.4) was solubilized by detergent from microsomal fraction of wounded Jerusalem-artichoke (Helianthus tuberosus L.) tubers and purified to electrophoretic homogeneity. The purification was achieved by two anion-exchange columns and by affinity chromatography on 2',5'-bisphosphoadenosine-Sepharose 4B. An Mr value of 82,000 was obtained by SDS/polyacrylamide-gel electrophoresis. The purified enzyme exhibited typical flavoprotein redox spectra and contained equimolar quantities of FAD and FMN. The purified enzyme followed Michaelis-Menten kinetics with Km values of 20 microM for NADPH and 6.3 microM for cytochrome c. In contrast, with NADH as substrate this enzyme exhibited biphasic kinetics with Km values ranging from 46 microM to 54 mM. Substrate saturation curves as a function of NADPH at fixed concentration of cytochrome c are compatible with a sequential type of substrate-addition mechanism. The enzyme was able to reconstitute cinnamate 4-hydroxylase activity when associated with partially purified tuber cytochrome P-450 and dilauroyl phosphatidylcholine in the presence of NADPH. Rabbit antibodies directed against plant NADPH-cytochrome c reductase affected only weakly NADH-sustained reduction of cytochrome c, but inhibited strongly NADPH-cytochrome c reductase and NADPH- or NADH-dependent cinnamate hydroxylase activities from Jerusalem-artichoke microsomal fraction.

show abstract

“…Whereas 3-epiecdysone (VII) was originally detected as a product of an enzyme, ecdysone 3-epimerase, from Manducu sextu midgut [3,4], several 3-epiecdysteroids have been isolated from that species [5,6]. 3-Epi-Zdeoxyecdysone as a phosphate conjugate has also been identified in developing eggs of Schisfocercn gregnria [7,8] and Locusta migraturia [9].…”

Section: Introductionmentioning

confidence: 99%

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

Gibson

Isaac

Dinan

et al. 1984

Arch Insect Biochem Physiol

View full text Add to dashboard Cite

The metabolism of [3H]-ecdysone has been investigated at times of low and high endogenous ecdysteroid titre, in early and late fifth-instar Schistocerca gregaria larvae, respectively. Ecdysone-3-acetateI 20-hydroxyecdysone, and 20,2&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 i s less, the major excreted rnatabolites 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 fifthinstar Locusta migratoria.Key words: ecdysone metabolism; Schistocerca; HPLC; ecdysteroid acids, acetates, phosphates INTRODUCTIONMetabolism, leading to inactivation and excretion of ecdysteroids, is an important factor in the regulation of circulating moulting hormone levels in Acknowledgments: We thank the S.E.R.C. for financial support and for a research studentship (to J.M.C.), Dr M.E. Rose and Mr M.C. Prescott for the mass spectra, and Professor T.W. Coodwin for his continued interest and encouragement.Received February 13, 1984; accepted March 28, 1984 Address reprint requests to Dr H.H. Rees 386Gibson et al insects. Known routes of ecdysteroid metabolism in insect systems include conversion into the 3-dehydro-, 3-epi-, 26-hydroxy-, and 3-acetate derivatives, the formation of polar ecdysteroid derivatives (conjugates and acids), as well as to a limited extent, C-20-C-22 side-chain cleavage (see [l] [16,19,201 and L. migratoriu [9,21,22]. In the latter case 22-adenosine monophosphate derivatives of ecdysteroids have also been reported [9,23].In the present paper, we report studies on the metabolism of [3H]-ecdysone in fifth-instar S. greguria. It has been reported that quantitative differences in ecdysteroid metabolism and excretion exist in fifth-instar Locusta migratoria at developmental stages when the endogenous hormone titre is low or high [24,25]. In view of this and in an attempt to idenhfy which metabolic processes undergo major quantitative changes during the instar suggesting their control during development, ecdysone metabolism was *Abbreviations: HPLC = high-performance liquid chromatograph...

show abstract

Ecdysone 3-epimerase from the midgut of Manduca sexta (L.)

Cited by 31 publications

References 16 publications

Expression of Housefly CYP6A1 and NADPH-Cytochrome P450 Reductase in Escherichia coli and Reconstitution of an Insecticide-Metabolizing P450 System

Expression of Housefly CYP6A1 and NADPH-Cytochrome P450 Reductase in Escherichia coli and Reconstitution of an Insecticide-Metabolizing P450 System

Purification and characterization of the NADPH-cytochrome P-450 (cytochrome c) reductase from higher-plant microsomal fraction

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

Contact Info

Product

Resources

About

Ecdysone 3-epimerase from the midgut of Manduca sexta (L.)

Cited by 31 publications

References 16 publications

Expression of Housefly CYP6A1 and NADPH-Cytochrome P450 Reductase in Escherichia coli and Reconstitution of an Insecticide-Metabolizing P450 System

Expression of Housefly CYP6A1 and NADPH-Cytochrome P450 Reductase in Escherichia coli and Reconstitution of an Insecticide-Metabolizing P450 System

Purification and characterization of the NADPH-cytochrome P-450 (cytochrome c) reductase from higher-plant microsomal fraction

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

Contact Info

Product

Resources

About

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