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The larval midgut of the tobacco hornworm, Manduca sexta, has high ecdysone 20-monooxygenase (EZOMO) activity, located both in the mitochondria and in the microsomes. The apparent kinetic parameters for E20MO in mitochondria and microsomes were determined. The K,s (for ecdysone) of the rnitochondrial and microsomal enzymes were 1.63 x 1 0-5 and 3.67 x 1 O-’ M, respectively. The V,,, was 82.7 prnol/min/rng protein for mitochondria and 32.0 pmol/min/mg protein for microsomes. Although the mitochondria1 E20MO has the higher V,,,, at physiological ecdysone concentrations (1 0-’ ~ 1 O-’ M) it is only one-eighth to one-tenth as active as the rnicrosornal enzyme. It is concluded that the rnicrosornal E20MO is the primary, if not the only, enzyme involved in ecdysone 20-hydroxylation in M. 5exta midgut. o 1996 Wey-Liss, Inc *

The activities of ecdysone oxidase (EO), 3-oxoecdysteroid 3α-reductase (3α-R), and 3-oxoecdysteroid 3β-reductase (3β-R) were determined for epidermis, hemolymph, and fat body of wandering fifth instar Manduca sexta larvae and for midguts of various developmental stages between 3 days after the last larval and 14 days after the pupal ecdysis. The larval midgut was the only organ showing substantial specific activities of EO and 3α-R, and both increased up to the seventh day after ecdysis. Hemolymph and fat body had only moderate to high 3β-R and low EO activites, and the epidermis did not contain significant activity of any of the enzymes. On the ninth day after the last larval ecdysis the larval midgut epithelium was replaced by a new pupal midgut epithelium. After this event only 3β-R was restored to high activities, whereas EO and 3α-R showed only low to marginal activities. It is concluded that only the larval midgut has a role in the inactivation of ecdysteroids by 3-epimerization. © 1993 Wiley-Liss, Inc. 1

Using high performance liquid chromatography in conjunction with radioimmunoassay and mass spectrometry, the major ecdysteroid of the solitary cactus bee, Diadasia rinconis, was determined to be 20-hydroxyecdysone, with lesser amounts of makisterone A. Another 28-carbon ecdysteroid thought to be the 24-epimer of makisterone A was also detected. The neutral sterols of Diadasia consisted primarily of 24-methylenecholesterol (92.2%) with lesser amounts of other C28 and C29 sterols. Cholesterol accounted for less than 0.1% of the total tissue sterols. The occurrence of 20-hydroxyecdysone in a phytophagous hymenopteran is discussed in relation to the low level of cholesterol encountered. © 1993 Wiley-Liss, Inc. 1

The 80,000g supernatant o larval midgut homogenates of the tobacco hornworm, Manduca sexta, was fractionated by affinity chromatography on Blue Sepharose CL-6B and by anion exchange chromatography on Q Sepharose. Both methods resolved one major 3-oxoecdysteroid 3α-reductase and three major 3-oxoecdysteroid 3β-reductases. The 3β-reducates reacted only with BADPH as cosubstrate. The 3α-reductase was active with both NADPH and NADH, and the NADPH/NADH activity ratio increased with the NaCl concentration (0–0.5 M) in the incubation mixtures. The 3-α-reductase and one of the 3-β-reductases showed very similar chromatographic properties, and their isoelectric points were 5.2 and 5.8, respectively. © 1992 Wiley-Liss, Inc.

An ecdysteroid RIA was used to determine the ecdysteroid titer in developing embryos of Dysdercus fasciatus and revealed that peak titer occurred approximately 120 h post-oviposition. Analysis of neutral sterols at this time indicated sitosterol to be the predominant neutral sterol with lesser amounts of campesterol. Embryonic sterols were highly reflective of the sterols found in the cotton seed diet upon which previous generations of the bugs had fed. Analysis of the embryonic extract for ecdysteroids indicated the presence of both makisterone A and the 29-carbon ecdysteroid makisterone C. Isolation of these compounds was accomplished by reversed-phase and silica HPLC in conjunction with RIA, and the identification of both compounds was confirmed by mass spectrometry. No ecdysone or 20-hydroxyecdysone was detected in the embryonic sample.

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.

The dual localization of ecdysone 20-monooxygenase in mitochondria and microsomes of Manduca sexta larval midgut was investigated. Cosubstrate requirements and response to osmolarity of the microsomal ecdysone 20-monooxygenase system were found to be different from those previously reported for the mitochondrial enzyme system. The microsomal monooxygenase utilized NADPH and, less efficiently, NADH as cosubstrates. NADPH and NADH effects were neither additive nor synergistic. NADPH yielded identical activities in isotonic and hypotonic incubations. Mitochondria and microsomes showed no synergistic interaction for ecdysone 20-hydroxylation. After washing of the mitochondria, a large proportion of their ecdysone 20-monooxygenase activity was lost. The extent of the loss was inversely correlated to the concentration of mitochondria in the incubation mixture. The addition of bovine serum albumin to the incubations (2 mg/ml) largely restored the original activities. The microsomal contamination in mitochondrial pellets after each of three successive washings was determined by measuring the activity of a microsomal marker enzyme, NADPH-cytochrome c reductase. At each step of the purification, the ecdysone 20-monooxgenase activity of the mitochondrial preparations far exceeded the activity attributable to the microsomal contamination. These results confirm the existence of two independent ecdysone 20-monooxygenase systems in the midgut of M. sexta larvae.

The subcellular distribution of four enzymes (glucose-6-phosphatase, phosphodiesterase I, NADPH-cytochrome c reductase, and p-nitroanisole O-demethylase) in the midgut of “wandering” fifth-instar larvae of the tobacco hornworm, Manduca sexta (L), was determined and the composition of mitochondrial and microsomal pellets was examined by electron microscopy. Most of the glucose-6-phosphatase activity and one-third of the phosphodiesterase I activity were found in the high-speed supernatant. NADPH-cytochrome c reductase activity was marginal and O-demethylase activity was undetectable in the supernatant. The highest specific activities for phosphodiesterase I, NADPH-cytochrome c reductase, and p-nitroanisole O-demethylase were measured in microsomes, but the relative specific activity of phosphodiesterase I was only half that obtained with the latter two enzymes. In all subcellular preparations the relative specific activities of NADPH-cytochrome c reductase and p-nitroanisole O-demethylase were closely correlated. It is concluded that glucose-6-phosphatase and phosphodiesterase I are not microsomal marker enzymes in the midgut, but the activities of NADPH-cytochrome c reductase and p-nitroanisole O-demethylase are quantitative measures of microsomal content.

In incubations with 80,000g supernatant of Manduca sexta midgut homogenates, [3H]ecdysone was converted to 3-[3H]epiecdysone and tritiumlabeled highly polar metabolites. C18 SEP-PAK cartridges were found suitable for the separation and purification of the free ecdysteroids and of the highly polar metabolites. Eighty to ninety percent of the metabolites were hydrolyzed by enzyme mixtures (mainly β-glucuronidase, sulphatase, and acid phosphatase) from molluscs, even when β-glucuronidase activity was completely inhibited by D-saccharic acid 1,4-lactone, or various human acid phosphatases (free of sulphatase activity). In each experiment, the hydrolysate contained a much higher proportion of 3-epiecydsone than the free (unconjugated) ecdysteroid fraction. [3H]ecdysone was not metabolized in anaerobic incubations of midgut supernatant that had been filtered through Sephadex G-25. Addition of 5 mM ATP and 5 mM Mg2+ restored the conjugate formation in incubations of Sephadex-filtered supernatant. Four ecdysone conjugates and two 3-epiecdysone conjugates were resolved by reversedphase ion-pair high-performance liquid chromatography. It is concluded that the midgut cytosol contains several ATP:ecdysteriod phosphotransferases. This is the first demonstration of the formation of ecdysteroid phosphoconjugates in a cell-free system.

The major ecdysteroid conjugate present in eggs (48–64 h old) of the tobacco hornworm has been purified by XAD-2 chromatography, C18 SEP-PAK separations, and ion suppression reversed-phase high-performance liquid chromatography. Enzymatic hydrolysis of the conjugate with acid phosphatase from human seminal fluid gave 26-hydroxyecdysone. The conjugate was identified as 26-hydroxyecdysone 26-phosphate by nuclear magnetic resonance and fast atom bombardment mass spectrometry. This compound is also the major conjugate of newly laid eggs (0–1 h old) of the tobacco hornworm. The role for ecdysteroid conjugates is discussed.