Archives of Insect Biochemistry and Physiology 12:201-218 (1 989) Ecdysone Oxidase and 3-Oxoecdysteroid Reductases in Manduca sexta Midgut: Kinet i c Parameters Gunter F. Weirich, Malcolm J. Thompson, and James A. Svoboda Insect Hormone Laboratory, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland 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 3wreductase(s), and one or more 3-oxoecdysteroid 3P-reductase(s). The reductases reacted at different velocities with NADH and NADPH. With NADH, 3a-reduction was the major reaction; with NADPH, 3P-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. Key words: 3-epimerizatior1, 3-dehydroecdysonef 3-epiecdysonef 3a-hydroxyecdysteroids, 3P-hydroxyecdysteroidsf NADH, NADPH, molting hormone inactivation INTRODUCTION 3-Epiecdysteroids (3a-hydroxyecdysteroids)have been isolated from several insect species [1,2]. Because of their low molting hormone activity [l-31 they are assumed to be inactivation products [2,4]. In vitro ecdysone 3-epimerization was first observed by Nigg et al. in incubations of ecdysone with midgut homog- enates of the tobacco hornworm, Manduca sexta L. [5]. The enzyme system for Acknowledgments: We thank Rosemary E. Hennessey and Lynda J. Liska for their dedicated technical assistance; and Drs. Govindan Bhaskaran, David J. Chitwood, and Herbert N. Nigg for critically reading the manuscript. Received August 18,1989; accepted October 25,1989. Some of the results reported in this paper have been presented in preliminary form at the Eighth Ecdysone Workshop, Marburg, Federal Republic of Germany, March 1987. Mention of a company name or proprietary product does not constitute an endorsement by the US. Department of Agriculture. Address reprint requests to Cunter F. Weirich, Insect Hormone Laboratory, Building467, BARC- East, Beltsville, MD 20705. 0 1989 Alan R. Liss, Inc. 202 Weirich et at. QH ?H 4H HO - HO - HO NAD(P)H HO 0 HO' Fig. 1. Proposed reaction sequence for the conversion of ecdysone (R = H) and 20-hydroxy- ecdysone (R = OH) to their 3(a)-epimers. this conversion was found to be located in the 85,OOOg supernatant and required NADH or NADPH for maximum activity. A two-step reaction was proposed, consisting of oxidation at carbon3 (requiring molecular oxygen) and NAD(P)H- dependent stereospecific reduction of the 3-0x0 intermediate to the 3-epimer [4,5] (Fig. 1). Ecdysone oxidase [6], the enzyme catalyzing the first of the two reactions, was purified from Culliphoru vicina pupae by Koolman and Karlson [7,8]. Ecdysone oxidase or its products have also been detected in several other species [3,9]. In further studies with M. sextu, Mayer et al. used a partially purified enzyme preparation from midgut to determine the kinetic parameters and to establish the oxygen requirement for the ecdysone 3-epimerization [4]; however, the postulated intermediate was not detected. Blais and Lafont [lo] succeeded in isolating 3-dehydro-20-hydroxyecdysoneand 3-epi-20-hydroxyecdysone from incubation mixtures of 20-hydroxyecdysone, NADPH, and enzyme prepara- tions from Pieris brussicue. These enzyme preparations also converted 3-dehydro- 20-hydroxyecd ysone to 20-hydroxyecdysone and 3-epi-20-hydroxyecdysone. The results demonstrated the existence of three cytosolic enzymes involved in the interconversion of 3P-hydroxysteroids, 3-oxosteroids, and 3a-hydroxysteroids: ecdysone oxidase and two different 3-oxosteroid reductases (3P-forming and 3a-forming, respectively). Milner and Rees [ll]obtained similar results with a dialyzed cytosolic enzyme preparation from Spodopteru littoralis midgut. M. sexta midgut cytosol, in addition to the enzymes of ecdysteroid 3-epimerization, also contains ecdysteroid phosphotransferases [ 121. The two enzyme systems compete for the ecdysteroid substrate, and their simulta- neous actions preclude exact measurements of their activities. However, the cosubstrates and cofactors necessary for the reactions (NADH or NADPH for 3-epimerization; ATP and Mg2+ for phosphoconjugation) can be eliminated by gel filtration on Sephadex G-25, and the individual enzyme activities can then be measured in the resulting partially purified enzyme preparation [12,13]. In this paper, we report evidence for the existence of ecdysone oxidase (ecdysone:oxygen3-0xidoreductase~ EC 1.1.3.17),3-oxoecdysteroid 3a-reductase (3a-hydroxyecdysteroid:NAD(P)+ oxidoreductase), and 3-oxoecdysteroid 3P-reductase (3P-hydroxyecdysteroid:NAD(P)+ oxidoreductase) in the mid- gut of M.sextu and present kinetic parameters for the enzymes. MATERIALS AND METHODS Chemicals Ecdysone (2P,3P, 14au,22R,25-pentahydroxy-5P-cholest-7-en-6-one)and 20- hydroxyecdysone (2P,3P, 14a,20R,22R,25-hexahydroxy-5~-cholest-7-en-6-one) Ecdysone Oxidase and Reductases 203 were obtained from Simes Pharmaceuticals (Milan, Italy) and Rhoto Pharma- ceutical Co. (Osaka, Japan), respectively. 3-Epiecdysone (2P,3a, 14a,22R,25- pentahydroxy-5p-cholest-7-en-6-one)[14], 3-dehydroecdysone (2P,14a,22R, 25-tetrahydroxy-5P-choles t-7-en-, 3,6-dione), and 3-dehydro-20-hydroxyecdysone (2~,14au,20R,22R,25-pentahydroxy-5~-cholest-7-en-3,6-dione)were synthesized [15] according to previously published procedures, and 3-epi-20-hydroxyecdy- sone (2P,3a, 14a,20R,22R,25-hexahydroxy-5~-cholest-7-en-6-one)was isolated from M. sextu meconium [ 161. NAD+ , grade V-C; NADP', monosodium salt; and Leuconostoc mesentemides Glc-6-P* dehydrogenase were obtained from Sigma Chemical Co. (St. Louis, MO); and Sephadex G-25 (fine), from Pharmacia LKB Biotechnology (Piscat- away, NJ). Enzyme Preparation Tobacco hornworms (M. sextu) were reared to the late fifth instar on artifi- cial diet [ 171. Midguts of "wandering" larvae were dissected and homogenized, and the 80,OOOg (3.6 x 10" rad2s- ) supernatant was prepared as described previously [13,18]. The supernatant was fractionated on Sephadex G-25 [12] equilibrated with 12.5 mM Tris-HC1 buffer, pH 7.5, containing 1.0 mM EDTA (buffer A). The combined protein (macromolecular) fractions (G-25 sup) were used for the incubations. Protein concentrations were determined according to Lowry et al. [19] with bovine serum albumin as standard. Incubations Ecdysone oxidase. Ecdysone (5-100 pM) or 20-hydroxyecdysone (38 pM) and G-25 sup (2.1-3.2 mg proteidml) were incubated for 2-4 h at 30°C (Dubnoff metabolic incubator, 90-100 oscillationsimin), in 0.5-2.0 ml of 30 mM potas- sium phosphate-10 mM Tris-HC1 buffer, pH 7.5, containing 1.0 mM EDTA (buffer B). After a 5-10 min equilibration period, the reactions were started by the addition of the ecdysteroid substrate, dissolved in 5-10 p1 methanol or 100 pl of buffer A. The incubations were stopped by the addition of 4.0 ml metha- nol. To obtain full oxidase activity it was necessary to use flat-bottom incuba- tion vials of 210 mm diameter to assure adequate agitation of the incubation mixtures and sufficient gas exchange. Incubations in 1.5 ml conical test tubes (Eppendorf) gave reduced rates of oxidation (80% of controls). 3-Oxoecdysteroid 3-reductases. Incubation mixtures contained 3-dehydro- ecdysone (2.5-100 pM) or 3-dehydro-20-hydroxyecdysone(38 pM) and G-25 sup in 0.5-2.0 ml of buffer 8. The concentrations of G-25 sup proteins in the incubation mixtures were 0.15-1.20 mg/ml with NADH as cosubstrate, or 0.01-0.12 mgiml with NADPH as cosubstrate. NADH or NADPH concentra- tions were 0.6 mM (obtained by addition of the appropriate amounts of NADt or NADP+, and a regenerating system consisting of 2.5 U of L. mesentemides Glc-6-P dehydrogenase/ml, and 6.0 mM Glc-6-P). L. mesentemides Glc-6-P dehy- *Abbreviations used: C-25 sup = rnacromolecular fraction of 80,OOOg supernatant of M. sexta rnidgut hornogenate, obtained by gel filtration on Sephadex G-25; = C-25 sup; Clc-6-P = glucose 6-phosphate. 204 Weirich et al. drogenase reacts with NAD+ and NADP+ at different rates, and the amount of enzyme added to the incubations was adjusted accordingly. The reaction mixtures were equilibrated in a Dubnoff metabolic incubator for 5-10 min (30 min for anaerobic incubations) at 30°C and 90-100 oscillations/ min. The reactions were started by addition of 3-dehydroecdysone or 3-dehydro- 20-hydroxyecdysone, dissolved in 50-100 pl of buffer A (for anaerobic incuba- tions, injected through the venting tube). Incubation times were 10-30 min, and the reactions were stopped by addition of 4.0 ml methanol. Anaerobic incubations. Vials (20 ml) containing the incubation mixtures were covered with rubber stoppers into which one gassing tube and one venting tube had been inserted. The incubation mixtures were purged with water- saturated nitrogen at approximately 100 ml/min. Again, a sufficient surface area was an important requirement for effective gas exchange. Infusion experiments. A solution of 3-dehydroecdysone (0.10 mM) in buffer A was infused into the incubation mixtures at a rate of 0.9 pl/min by a Sage syringe pump (Orion Research, Boston, MA). For anaerobic incubations, the tubing delivering the solution was inserted through the venting tube in the vial stopper. Kinetic experiments. The protein concentrations were adjusted to assure lin- earity of the reactions for the duration of the incubations (10 min), and the reaction rates were proportional to the amounts of protein added. Extraction and Purification 3-Dehydroecdysone and 3-dehydro-20-hydroxyecdysoneeach form an unknown decomposition product of lower polarity than the parent compound (see Fig. 2) [lo]. Only marginal amounts of the decomposition product(s) were formed in methanolic solutions or in solutions of the 3-oxoecdysteroids in buffer A even during prolonged storage under refrigeration.