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ABSTRACT: The tritium-labeled bis-norleucine analog of Helicoverpa zea pheromone biosynthesis-activating neuropeptide ([3H]NLPBAN) was incubated in vitro with hemolymph from Manduca sexta or H. zea adult females. The incubations resulted in the formation of several tritium-labeled degradation products. At a [3H]NLPBAN concentration of 0.9 microM the degradation proceeded at a very slow but physiologically plausible rate (2-10 fmol/min/microliters hemolymph). The primary [3H]NLPBAN degradation reaction in M. sexta hemolymph was not inhibited by 20 microM leupeptin, 0.1 mM amastatin, 1 mM EDTA, 1 mM EGTA, 1 mM 1,10-phenanthroline, or 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride; but secondary reactions may have been affected, as some of the inhibitors changed the radio-HPLC profile of the degradation products. It is concluded that hemolymph of M. sexta and H. zea contains peptidase(s) capable of inactivating circulating PBAN.
Experientia 10/1995; 51(9-10):961-6.
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ABSTRACT: Larvae of Drosophila melanogaster were reared aseptically on defined diets containing either cholesterol, campesterol or sitosterol as the only dietary sterol. Sterol analyses of pupae revealed that insects reared on campesterol and sitosterol diets contained 3.3 and 8.1% cholesterol, indicative of an ability to accumulate this sterol. Ecdysone and 20-hydroxyecdysone were the predominant ecdysteroids in insects from all diet studies, though makisterone A was detected in pupae reared on campesterol and sitosterol.
Insect Biochemistry and Molecular Biology 07/1995; 25(6):709-12. · 3.25 Impact Factor
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ABSTRACT: A number of intermediates involved in the dealkylation and conversion of the major C28 and C29 phytosterols to cholesterol in insects were first isolated and identified in studies with the tobacco hornworm, Manduca sexta, carried out in our laboratory. We also investigated the effects of a variety of known sterol metabolism inhibitors in Manduca, particularly those affecting the delta 24-sterol reductase enzyme, and synthesized and tested a number of new inhibitors as well. In-depth studies of ecdysteroids in Manduca during embryogenesis and during pupal-adult development provided new information on molting hormone content, biosynthesis, and metabolism. In addition, this insect has been utilized in the study of three specific enzyme systems of ecdysteroid metabolism, namely 20-monooxygenase, 3-epimerase, and phosphotransferase, which are critical to activation and deactivation of molting hormones in insects.
Lipids 04/1995; 30(3):263-7. · 2.13 Impact Factor
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Analytical Biochemistry 02/1994; 216(1):228-32. · 3.00 Impact Factor
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ABSTRACT: Following injection into Manduca sexta (L.) female pupae (day 16), [14C]cholesterol was converted to a C21 steroid conjugate, 5-[14C]pregnen-3 beta,20 beta-diol glucoside. The conjugate was isolated from ovaries and eggs and contained three glucose units at least one of which is attached to C-20. The distribution of the other two glucose units remains to be determined. Other than the dealkylation of C-24 alkane or alkene substituents, side-chain cleavage of sterols is uncommon to insects. Here we report the first definitive proof of the biosynthesis of a C21 steroid conjugate from cholesterol in an insect species. The capability of M. sexta to so readily convert cholesterol to a C21 steroid suggests a physiological role for 5-pregnen-3 beta,20 beta-diol in this species.
Journal of Biological Chemistry 01/1986; 260(29):15410-2. · 4.77 Impact Factor
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ABSTRACT: Ecdysteroids of ovaries and newly-laid eggs (0- to 1-hour-old) of the tobacco hornworm are present mainly as conjugates (greater than 95%). Newly-laid eggs contain ecdysteroid conjugates equivalent to 21 micrograms of 26-hydroxyecdysone and 0.73 micrograms of ecdysone per gram of eggs. These levels are similar in ovaries of 93-hour-old adult females. In 1- to 18-hour-old eggs more than 63% of the ecdysteroids exist in the free form and the proportion is similar in 48- to 64-hour-old eggs. The ratio of 26-hydroxyecdysone to ecdysone in the conjugated form remains constant during oocyte maturation and embryogenesis. Though 26-hydroxyecdysone is without molting hormone activity in the house fly assay, the exceptionally high concentration of 26-hydroxyecdysone conjugate(s) in ovaries and newly-laid eggs, together with the fact that it is being released during embryogenesis, indicate some physiological role for 26-hydroxyecdysone.
Steroids 04/1984; 43(3):333-41. · 2.83 Impact Factor
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ABSTRACT: Ecdysone 3-epimerase was partially purified by ammonium sulfate fractionation from the 100,000 g supernate of Manduca sexta midguts. The enzyme converts ecdysone and 20-hydroxyecdysone to their respective 3-epimers, requires NADH or NADPH and O2 for this reaction, and has the following kinetic parameters: for ecdysone, Km = 17.0 +/- 1.4 microM, Vmax = 110.6 +/- 14.6 pmol min-1 mg-1; for 20-hydroxyecdysone, Km = 47.3 +/- 7.5 microM, Vmax = 131.0 +/- 3.5 pmol min-1 mg-1: for NADPH, Km = 85.4 +/- 10.6 microM; for NADH, Km = 51.3 +/- 1.3 microM. The reaction is irreversible and can be inhibited by various ecdysteroids.
Steroids 12/1979; 34(5):555-62. · 2.83 Impact Factor
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ABSTRACT: The conversion of alpha-ecdysone to 20-hydroxyecdysone in the midgut of Manduca sexta (L.) was found to be catalyzed by a mitochondrial, cytochrome P450-mediated monooxygenase. The reaction required oxygen and was inhibited by the presence of carbon monoxide. Tricarboxylic acid cycle intermediates, such as succinate, malate, and isocitrate, supported the hydroxylation as did NADPH, NADH, ATP, and ADP. Temperature and pH optima were 30 degrees C and 8.5, respectively. The apparent Km and V values for the ecdysone 20-hydroxylase were 18.3 +/- 6.8 micronM and 46.6 +/- 14.2 pmol per min per mg protein. The midgut mitochondria were found to contain malate dehydrogenase and NAD(P) transhydrogenase. The presence of these enzymes suggests that the tricarboxylic acid cycle intermediates and NADH support the ecdysone hydroxylation indirectly by providing NADPH for the cytochrome P450 system. The content of cytochromes a + a3, b, c + c1, and P450 in midgut mitochondria was determined.
Hoppe-Seyler's Zeitschrift für physiologische Chemie 11/1978; 359(10):1247-57.
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ABSTRACT: 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 K(m)s (for ecdysone) of the mitochondrial and microsomal enzymes were 1.63 X 10(-5) and 3.67 X 10(-7) M, respectively. The V(max) 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 V(max), 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.
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ABSTRACT: Ecdysone and 20-hydroxyecdysone are converted to their 3-epimers by enzymes in the midgut cytosol of Manduca sexta larvae. A partially purified cytosol preparation has been used to analyze the nature of and the interaction between these enzymes. The cytosol was shown to contain ecdysone oxidase, one or more 3-oxoecdysteroid 3α-reductase(s), and one or more 3-oxoecdysteroid 3β-reductase(s). The reductases reacted at different velocities with NADH and NADPH. With NADH, 3α-reduction was the major reaction; with NADPH, 3β-reduction was the major reaction. The apparent kinetic parameters for the enzymes support the assumed two-step mechanism for the 3-epimerization with a 3-oxoecdysteroid as intermediate.
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ABSTRACT: Larvae of Drosophila melanogaster were reared aseptically on defined diets containing either cholesterol, campesterol or sitosterol as the only dietary sterol. Sterol analyses of pupae revealed that insects reared on campesterol and sitosterol diets contained 3.3 and 8.1% cholesterol, indicative of an ability to accumulate this sterol. Ecdysone and 20-hydroxyecdysone were the predominant eedysteroids in insects from all diet studies, though makisterone A was detected in pupae reared on campesterol and sitosterol.
Insect Biochemistry and Molecular Biology.
