Ecdysone Oxidase and 3-Oxoecdysteroid Reductases in Manduca Sexta Midgut: Kinet I C Parameters

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-hydroxyecdysone (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 preparations 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 midgut 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-hydroxyecdysone (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 methanol. To obtain full oxidase activity it was necessary to use flat-bottom incubation 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-dehydroecdysone (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 concentrations 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 incubations, 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. However, complete drying of the samples in methanol-buffer mixtures increased the proportions of the decomposition products substantially. We therefore used the following proto- col for sample processing. Water was added to the samples to bring the water content to 2.0 ml and the total volume to 6.0 ml (4.0 ml of methanol was added at the end of the incubations). The protein was sedimented by a 15 min centrifugation at 20,OOOg. The supernatants were partially evaporated in a Speed Vac concentrator (Savant Instruments, Farmingdale, NY) at room temperature to between 1.8 and 2.0 ml with a concomitant reduction of the methanol content to about 10%. The samples were then purified on CISSEP-PAK cartridges as described previously [12,13]. The 60% methanol (free ecdysteroid) fractions were dried in the Speed Vac concentrator at room temperature, redissolved in 50-100 p1 methanol and analyzed by HPLC. HPLC Most of the experimental samples were analyzed by reversed-phase HPLC on a C18 Radial Compression column (10 cm x 5.0 mm, 10 pm particle size; Waters, Milford, MA) at 35°C with 15518%acetonitrile in water as mobile phase, and the column was rinsed for 10 min with 50% acetonitrile in water after each analysis [13]. Ecdysteroids were quantified by peak area integration of Ecdysone Oxidase and Reductases 205 the 254 nm absorbance peaks (Waters Data Module 730). To confirm the identity of the reaction products, some samples were also analyzed by normal- phase HPLC on an Ultrasphere Si column (25 cm x 4.6 mm, 5 km particle size; Beckman, Berkeley, CA) at 35°C with dichloromethane/2-propanol/wateras mobile phase [lo] (for proportions see Figs. 2 and 5).

Calculations of Apparent Kinetic Parameters The reciprocals of substrate concentrations and velocities were subjected to linear regression analysis and a two-tailed t-test. Only sets of data that resulted in a confidence limit of ~99%were evaluated and reported in Figures 6 and 7.

RESULTS Ecdysone Oxidase When incubated with postmicrosomal supernatant of M. sextu midgut, ecdysone is converted to 3-epiecdysone even if no NADH or NADPH is added to the incubation mixture [12]. The supernatant apparently contains the cosubstrate(s) required for the reaction. The presumed intermediate, 3-dehydroecdysone, however, has not been found in these incubation mixtures. In the present study, the postmicrosomal supernatant was filtered through a Sephadex G-25 column to eliminate endogenous NADH, NADPH, and other low-molecular-weight components [12]. Incubations of this enzyme preparation (G-25 sup) with ecdysone did not yield 3-epiecdysone, but 3-dehydroecdysone instead (Fig. 2A,B). With 20-hydroxyecdysone as substrate, the product of the incubation was 3-dehydro-20-hydroxyecdysone(Fig. 2C,D). The reaction required oxygen. In anaerobic incubations or incubations with boiled G-25 sup, ecdysone was not converted to either 3-dehydroecdysone or 3-epiecdysone. These results indicated that M. sextu midgut contains an ecdysone oxidase and provided experimental proof for the first part of the reaction sequence shown in Figure 1.

Ecdysone Oxidation and 3-Epimerization The failure to detect 3-dehydroecdysone in incubations of ecdysone with complete postmicrosomal supernatant suggested that 3-dehydroecdysone was reduced to 3-epiecdysone as rapidly as it was formed. To test this assumption, G-25 sup and ecdysone were incubated with or without the addition of reduced cosubstrates. Figure 3 shows the conversion of ecdysone to 3-dehydroecdysone in the absence of reduced cosubstrates, to 3-epiecdysone with the added NADPH-regenerating system, and to 3-epiecdysone and a small amount of 3-dehydroecdysone with the added NADH-regenerating system. The yield of 3-epiecdysone with either cosubstrate was lower than the yield of 3-dehydroecdysone without the cosubstrate. A small amount of 3-dehydroecdysone was found in NADH-containing incubations but not in NADPH-containing incubations. The results showed that none or very little of the 3-dehydroecdysone formed by ecdysone oxidation remained unchanged when NADH or NADPH was present in the incubation mixtures, but they also showed that not all of the missing 3-dehydroecdysone was reduced to 3-epiecdysone. 206 Weirich et al.

(A) 30I i i E'

,I I I I I I I I 0 10 rnin 20 30 0 10 20 30 rnin

3Df0 20E', I

pI0

sc I I LI 1 0 10 22 0 10 20 30 min rnin

Fig. 2. Ecdysone Oxidase and Reductases 207

p 3D / /

0 120 mi" 6o t Fig. 3. Conversion of ecdysone by (2-25 sup enzymes with or without added reduced cosubstrates. Ecdysone (50 FM) and G-25 sup (2.6 mg protein) were incubated at 30°C in 1.O ml of buffer B without added cosubstrate (O---O), with 0.6 mM NADH and regenerating system (A-.-A,O- -O),orwith0.6mM NADPH and regeneratingsystem [A-A). E',3-epiecdysone; 3D, 3-dehydroecdysone. Ordinate: nmol/mg protein.

3-Oxoecdysteroid 3a-Reductase and 3-Oxoecdysteroid 3P-Reductase To further explore this relationship, G-25 sup was incubated anaerobically (to prevent reoxidation of reduced ecdysteroids) with 3-dehydroecdysoneand NADH or NADPH (Fig. 4). With either one of these reduced cosubstrates, 3-dehydroecdysone was reduced very rapidly. The reaction was completely suppressed by omission of reduced cosubstrates or by boiling of G-25 sup. The rates of the 3-dehydroecdysone reductions were much higher than those observed for the ecdysone oxidation at equal substrate concentration (cf. Fig. 3). The identity of the enzymatic reduction products obtained from incubations of 3-dehydroecdysone or 3-dehydro-20-hydroxyecdysoneand NAD(P)H with G-25 sup was verified by comparison of retention times with those of

Fig. 2. HPLC analyses of incubation products of ecdysone or 20-hydroxyecdysone and C-25 sup. Ecdysone (50 FM; A,B) or20-hydroxyecdysone (38 pM; C,D) were incubated with G-25 sup (3.2 mg protein) in 1.0 ml of buffer B for 4 h at 30°C. Incubation mixtures were extracted and purified as described in Materials and Methods and analyzed by HPLC on a Waters Radial Compression CI8coIumn (10 cm x 5 mm, 10 p.m particle size) (A,C), or a Beckman Ultrasphere Si column (25 cm x 4.6 mm, 5 Frn particle size) (B,D) at 35°C. The solvent systems were: 16% acetonitrile in water (A), 15% acetonitrile in water (C), both followed by a 10 min column flush with 50% acetonitrile in water (not shown); CHzCLz/2-propanol/water, 125/25/2 (B), CH2CL2/ 2-propanol/water, 12513112.5 (D). Flow rate for all systems was 1.O ml/min. Arrows mark elution times for ecdysone (E), 3-epiecdysone (E'), 3-dehydroecdysone (3D), 20-hydroxyecdysone (20E), 3-epi-20-hydroxyecdysone (20E'), and 3-dehydro-20-hydroxyecdysone (3D20) in the corresponding system. The peaks marked X represent unknown products of 3-dehydroecdysone or 3-dehydro-20-hydroxyecdysone decomposition during extraction and purification of samples. UVabsorbance was monitored at 254 nm; bars indicate absorbance units. 208 Weirich et al.

-g

40 / / 150 / / I/ I 20 / I/ / I 50

Fig. 4. Conversion of 3-dehydroecdysone by G-25 sup enzymes with NADH or NADPH. 3-Dehydroecdysone (50 pM) and G-25 sup (A, 0.60 mg protein; B, 0.12 mg protein) were incubated anaerobically in 1.O ml of buffer B at 30°C. A: 0.6 mM NADH and regenerating system; B: 0.6 mM NADPH and regenerating system. E, ecdysone; E', 3-epiecdysone. Ordinate: nmolimg protein.

ecdysteroid standards in reversed-phase HPLC and in normal-phase HPLC (Fig. 5). These experiments showed that 3-dehydroecdysteroids can indeed be converted to 3-epiecdysteroids by an enzyme(s) in M. sexta midgut as postulated by Nigg et al. [5], but they also showed a conversion of 3-dehydroecdysone to ecdysone, a reversal of the ecdysone oxidation. Thus, M. sexta midgut contains both 3-oxoecdysteroid 3a-reductase(s) and 3-oxoecdysteroid 3P-reductase(s). Both types of reductions can be supported by either NADH or NADPH as cosubstrate. With NADH, mostly 3a-hydroxyecdysteroids were formed; but with NADPH, 3P-ecdysteroids were the major product (Fig. 4, Table 1).Apparently, NADH is a better cosubstrate for the 3a-reductase(s) than NADPH, and NADPH works better with 3@-reductase(s).The activity ratio between the a- and P-reductases was also affected by the nature of the ecdysteroid substrate (3-dehydroecdysone vs. 3-dehydro-20-hydroxyecdysone; Table 1). The 'difference between the total 3-dehydroecdysone formation and the 3-epiecdysone formation (from ecdysone as added substrate) shown in Figure 3 can be attributed to the reduction of some 3-dehydroecdysone to ecdysone. The ratios of 3a-reduction to 3P-reduction calculated for this and similar experiments were 80/20 to 100/0 with NADH as cosubstrate, and 45/55 to 60/40 with NADPH as cosubstrate. In experiments with 3-dehydroecdysone as added sub-

Fig. 5. HPLC analyses of metabolites obtained from incubations of 3-oxoecdysteroids and reduced cosubstrates with G-25 sup. A,B: 3-Dehydroecdysone (50 pM) and C-25 sup (1.2 mg protein) were incubated anaerobically with 0.6 mM NADH and regenerating system in 1.0 ml of buffer B for 30 min at 30°C. C,D: 3-Dehydro-20-hydroxyecdysone (38 pM) and G-25 sup (0.8 mg protein) were similarly incubated with 0.6 mM NADPH and regenerating system. Incuba- tion mixtures were extracted, purified, and analyzed as in Figure 2, except for the solvent system of D, which was CH2CL,/2-propanol/water, 125/35/2.8. Symbols and abbreviations as in Figure 2. Ecdysone Oxidase and Reductases 209

(A)

30 7' I f

1, I I I I I I I 0 20 30 0 20 30 lo min lo min

3D20 I

(D) 20E

I I

I I I I 20 30 0 10 20 0 lo min rnin

Fig. 5. 210 Weirich et al.

TABLE 1. Reduction of 3-Dehydroecdysone and 3-Dehydro-20-hydroxyecdysoneby G-25 sup Enzymes* Concentration NADH NADPH ((*MI (a)” (49” 3-Dehydroecd ysone 50 76124 6/94 3-De hydro-20-hydroxyecd ysone 38 90110 21179 *Incubation mixtures contained G-25 sup (1.2 mg protein for incubations with NADH, 0.8 mg protein for incubations with NADPH) and 0.6 mM NADH or 0.6 mM NADPH, plus regenerating system, in 1.O ml of buffer €3, and were incubated for 30 min at 30°C. “nIP,ratio of Sa-hydroxy- to 3P-hydroxyecdysteroid. strate (Fig. 4 and similar experiments), the 3a/3P ratios obtained with NADH as cosubstrate were similar (76/24 to 96/4), but the proportions of 3a-hydroxysteroid obtained with NADPH were much lower (3a/3P,3/97 to 16/84) than in the experiments using ecdysone as added substrate. This difference in the ratios of 3a-reduction to 3p-reduction could have several causes:

1. 3P-Reduction of 3-dehydroecdysone could be subject to product inhibition by ecdysone. The 3P-reduction would be inhibited in incubation mixtures containing ecdysone as thhe initial substrate, and as a result, the 3d3P ratio would be changed in favor of 3a. 2. 3a-Reduction and 3P-reduction could be affected differently by the substrate concentration. When 3-dehydroecdysone was the added substrate, it was present in rather high concentrations, but when ecdysone was the added substrate, 3-dehydroecdysone was produced by the ecdysone oxidase only in very small quantities. 3. Oxygen (present in incubations with ecdysone, absent from incubations with 3-dehydroecdysone) could affect 3a-reductase and 3P-reductase differently.

To test these possible causes, four types of incubations were set up with the same G-25 sup preparation, and after completion, the proportions of 3a-hydroxysteroids and 3P-hydroxysteroids were determined: a) 10 pM 3-dehydroecdysone (high substrate concentration) and NADH or NADPH, b) continuous infusion of 3-dehydroecdysone at a slow rate (low substrate concentration) and NADH or NADPH, c) 50 pM ecdysone (as a source for 3-dehydroecdysone at low concentrations) and NADH or NADPH, and d) 50 p.M ecdysone without reduced cosubstrates (to determine the rate of ecdysone oxidation). The results (Table 2) showed that low 3-dehydroecdysone concentrations (obtained by infusion or by ecdysone oxidation) yielded higher proportions of 3-epiecdysone than incubations with a high 3-dehydroecdysone concentration when NADPH was used as cosubstrate. To a lesser extent, that was also true for incubations with NADH as cosubstrate, but the 3-epiecdysone yields were within the over- lapping ranges found earlier for 3-dehydroecdysone and ecdysone incubations. The incubations involving 3-dehydroecdysone infusion and those utilizing ecdysone oxidation as source of 3-dehydroecdysone yielded almost identical ratios of 3a- to 3P-reduction. The presence of a large excess of ecdysone in the latter incubations did not affect the relative rates of the 3-dehydroecdysone reduc- Ecdysone Oxidase and Reductases 21 1

TABLE 2. 3-Dehydroecdysone Reduction by G-25 sup Enzymes at High and Low Substrate Concentrations*

~ NADH NADPH ~~ 3D Supply E E' E E' Experiment (*M pmollminlmg (pmollminlmg) (alp) (pmollminlmg) (alp) 1 Air 10 Add 79121 18182 Air 40 Inf 2.4 30.4 9317 19.6 20.3 51149 Air 42 Eoxid 1.9 35.1 9515 17.9 23.6 57143 2 Air 10 Add 79121 28172 Air 30 Inf 2.4 23.4 9119 14.7 14.0 49151 Air 45 Eoxid 4.4 37.8 90110 24.6 20.5 45155 3 Air 10 Add 18182 Nitrogen 10 Add 15185 Air 35 Inf 19.2 13.5 41159 Nitrogen 35 Inf 20.5 12.6 38162

*Incubation mixtures contained G-25 sup (2.1-3.1 mg protein), and 0.6 mM NADH or 0.6 mM NADPH, plus regenerating system, in 1 .O ml of buffer B. 3-Dehydroecdysone (3D) was supplied to incubation mixtures at the indicated concentrations or rates, either by initial addition (Add), continuous infusion (Inf), or by enzymatic oxidation of ecdysone (50 pM) at the indicated rates (E oxid). The ecdysteroid concentrations were selected to obtain reduction of about 10 nmol 3-dehydroecdysone in each incubation. E, ecdysone; E', 3-epiecdysone; alp, ratio of 3a-reduction to 3p-reduction, determined after 120 min incubation for 'Inf and 'E oxid', after 10 min for 'Add' incubations. All 3D was reduced during the 10 min 'Add' incubations, and accordingly the rates could not be determined. tions, and product inhibition did not occur. The presence or absence of oxygen did not affect the 3a/3P ratios either (Table 2, experiment 3). In conclusion, these results ruled out assumptions 1and 3 and suggested that the 3-dehydroecdysone concentration has a strong effect on the ratio of 3a- to 3P-reduction with NADPH (assumption 2). Kinetic Parameters To further characterize the ecdysone oxidase and 3-oxoecdysteroid 3- reductases of M. sextu midgut, the apparent kinetic parameters were determined. By incubation of G-25 sup and 3-dehydroecdysone with either NADH or NADPH, four sets of kinetic parameters were obtained, corresponding to 3P-reduction with NADH, 3a-reduction with NADH, 3P-reduction with NADPH, and 3a-reduction with NADPH. Although it is not clear at present whether these reactions are catalyzed by one 3a-reductase and one 3@-reductase, each displaying different kinetic parameters with each of the two cosubstrates, or by four different cosubstrate-specific reductases, for the sake of simplicity they will be referred to as four individual reductases. Figure 6 shows exam- ples of velocity-vs.-substrate-concentrationand double reciprocal plots for each of the five enzymes. Figure 7 presents the apparent kinetic constants as means ( ? SD) of several determinations. The high standard deviations for some of the apparent kmetic parameters probably reflect variations between different batches 21 2 Weirich et al. A

0.1 0 0.1 0.2 i/CS] (#MI-'

0.1 0 E ..-I= .E 0 0.05 E

0.1 0 0.1 1/[S] (rM1-I0.2

0.1 0 0.1 0.2 Fig. 6. 1/[S3 (gM)-' Ecdysone Oxidase and Reductases 21 3

3.0t 7 - 2.0 0. P' a E .-.E -.E- 0 E Y > -1.0 .r

0 50 100 cd (pM)

I 1 0 0.1 0.2 1/[Sl bW-'

0.4 0.2 0 0.2 0.4 1/cSl (uM)-' Fig. 6. Velocity-vs.-substrate-concentration plots and double reciprocal plots for ecdysone oxidase (A), NADH-dependent 3-oxoecdysteroid 3P-reductase (B), NADH-dependent 3-oxoecdysteroid 3a-reductase (C), NADPH-dependent 3-oxoecdysteroid 3P-reductase (D),and NADPH- dependent 3-oxoecdysteroid 3a-reductase (E) of M. sexta midgut cytosol. Incubation mixtures contained the substrates ecdysone (A) or 3-dehydroecdysone (B-E); 0.6 mM of NADH (B,C) or NADPH (D,E), plus regenerating system; and G-25 sup to give a protein concentration of 2.25 mgiml (A), 0.15-0.28 mg/ml (B,C), or 0.010-0.065 mglml (D,E); in 1.0-2.0 ml of buffer B, and were incubated anaerobically for 10 min (B-E) or 60 min (A) at 30°C. of animals. A separate G-25 sup preparation was used for each of the two-to- four determinations of the parameters. Except for the NADPH-dependent 3a-reductase, the Michaelis constants for the ecdysteroid substrates were all in the 10-to-30 pM range. The K, for NADPH-dependent 3a-reductase was found to be about 2 pM, and the enzyme showed apparent substrate inhibition above 10 pM. Although data showing such inhibition were not used for the computations, the apparent kinetic parameters are somewhat uncertain because of this complication. The K, for ecdysone oxidase was about the same 214 Weirich et al.

4 E > 3D Ecdysone oxidase

K, 13.3 k1.8 pM(2)

V,,, 0.047 f 0.000 1 nmol(min mg protein)-'

k 3.55 +_ 0.50 pmol(min pM mg protein)-'

NADH NADH E 3D A E' -3p - Reductase 3 a -Reductase

Km 15.5 2 3.2(3) Km 30.7 2 16.2(4)

Vmax 0.18 k 0.04 Vm,, 4.06 f 2.20

k 11.8 2 0.9 k 132 214

NADPH NADPH i- E i- 3D A E' 3 p -Reductase 3 a -Reductase

K, 17.7 k 10.3(4) K, 2.12 +0.42(2)

Vma, 10.7 2 3.3 Vmax 0.64 +_ 0.04

k 619 2182 k 304k44

Fig. 7. Apparent kinetic parameters of ecdysone oxidase, NADH-dependent 3-oxoecdysteroid 3P-reductase, NADH-dependent3-oxoecdysteroid 3a-reductase, NADPH-dependent 3-oxoecdysteroid 3p-reductase, and NADPH-dependent 3-oxoecdysteroid 3a-reductase. Means 2 SD, number of experiments in parenthesis. k = V,,,IK,, first order rate constants for very low substrate concentrations (<

as that previously determined for the complete epimerization sequence with another partially purified enzyme preparation from M. sexta midgut (17.0 2 1.4 FM) [4]. The maximum velocities differed widely; and the V,,, for ecdysone oxidase, as expected, was the lowest. The high V,,, for the 3a-reduction with NADH and for the 3P-reduction with NADPH and the low V,,, for the other two reductases agreed fairly well with the progress curves shown in Figure 4. To compare the rates of these reactions at physiological substrate concentrations (<

v = [S]V,,,/K,.

Comparisons of the V,,, and k suggested that the 3-dehydroecdysone concentration has only a minor effect on the 3d3P reduction ratio with NADH as cosubstrate. With NADPH as cosubstrate, however, the 3a/3P reduction ratio should increase from about 7/93 to about 30/70 when the 3-dehydroecdysone Ecdysone Oxidase and Reductases 21 5 concentration is lowered from very high (>K,) to very low levels (<

DISCUSSION The present study was undertaken to investigate the mechanism of the epimerization of 3p-hydroxyecdysteroidsin M. sexta midgut. Our results showed that the second step of the proposed two-step conversion can be blocked by removal of reduced nicotinamide adenine dinucleotides (and other small mol- ecules) from the cytosol by gel permeation chromatography on Sephadex G-25. With this partially purified enzyme preparation (G-25 sup), the reaction pro- ceeded only to the %OX0 intermediate, and the reaction required oxygen, as had previously been shown for the complete epimerization sequence [4,12]. These observations indicated that the midgut of M. sexta contains a cytosolic ecdysone oxidase. To test the second step of the proposed sequence, 3-dehydroecdysone or 3-dehydro-20-hydroxyecdysonewere incubated with G-25 sup and NADH or NADPH. The results showed that M. sexta cytosol not only contains 3-oxoecdysteroid 3w-reductase(s), implied in the proposed two-step reaction scheme, but also 3-oxoecdysteroid 3p-reductase(s), catalyzing the reconver- sion of the %OX0 intermediate to the original 3P-ecdysteroid. 3-Oxoecdysteroids are apparently not only intermediates in the conversion of active molting hormones to the much less active 3-epimers, but can also be (re)converted to the active 3P-hydroxyecdysteroids [lo, 111. According to a recent report, 2-dehydroecdysone and/or 3-dehydroecdysone are the products released by the prothoracic glands of M. sexta in vitro and can be converted to ecdysone by hemolymph-borne reductase(s) [20]. The (mid)guts of M. sexta, P. brassicae, and S. liftoralis have similar sets of enzymes involved in the interconversion of 3P-hydroxyecdysteroids, 3-0x0- ecdysteroids, and 3a-hydroxyecdysteroids [ 10,11,21], but there are probably differences between species in the properties, interaction, and control of these enzymes. The reductases in M. sexta midgut produced mainly 3-epiecdysone when NADH was added as cosubstrate, mainly ecdysone when NADPH was added as cosubstrate. The reverse has been found for S. littoralis [ll].The partially purified enzyme preparation obtained earlier from M. sexta [4] had shown about equal rates of ecdysone 3-epimerization with either NADH or NADPH. This differencefrom our current results may have been caused by partial elimi- nation or inactivation of 3P-reductase(s) during storage (at - 20°C)and/or ammo- nium sulfate fractionation of the cytosol. The apparent kinetic parameters of the M. sexta enzymes (Fig. 7) suggest the mode of their interaction. The five enzymes do not differ much in their affinity for their ecdysteroid substrates. The K, of four of the five enzymes are very similar (13-31 pM), and the K, for the NADPH-dependent 3a-reductase is only about one order of magnitude lower. The V,,,, on the other hand, differ widely. The V,,, of the four reductases exceed the V,,, of oxidase 4-228 times. However, the V,,, are unlikely to determine the quantitative relations of the enzymatic reactions in the midgut cells, because the substrate concentrations (especially those of 3-dehydroecdysone) are probably far below 216 Weirich et al. those required for V,,,. The comparison of the first order rate constants (k) should provide a more realistic estimation of the interaction. The first order rate constants of the four reductases exceed that of ecdysone oxidase 3-190 times. Thus, the ecdysone oxidase is the rate-limiting enzyme both at opti- mum and at physiological substrate concentrations. On the basis of their very high rate constants, the NADPH-dependent reductases would seem to be the dominating reductases. However, with NADPH, more than two-thirds of the 3-dehydroecdysone would be reconverted to ecdysone in what appears to be a futile cycle, and only less than one-third would be converted to 3-epiecdysone. Thus, despite their apparent high efficiency, they would not be very suitable for an efficient inactivation of ecdysone (or 20-hydroxyecdysone). With NADH as cosubstrate, 3a-reduction would exceed 3P-reduction more than 10-fold, and therefore, although functioning at a lower rate than the NADPH-supported reactions, the NADH-supported reductions would provide for an effective 3a-reduction of 3-dehydroecdysone at a rate exceeding the rate of ecdysone oxidation 37 times. The apparent kinetic parameters have been determined at a uniform NAD(P)H concentration of 0.60 mM. We do not know at present how these parameters would be affected by different cosubstrate concentrations, and what these concentrations are in midgut cells. Perhaps the balance between ecdysone recycling and ecdysone 3-epimerization is controlled via the balance between NADH and NADPH in the cells. The answer to these questions will have to await the analysis of NADH and NADPH concentrations in the tissue. The apparent kinetic parameters of the reductases could reflect the presence of as many as four different enzymes or as few as two enzymes, one 3a-reductase and one 3P-reductase, functioning at different levels with NADH and NADPH. To answer this and other open questions, work on the isolation of the enzymes is currently underway. The results reported here show that the postulated enzyme activities [4] are indeed present in M. sextu midgut cytosol, as has been shown for P. brassicae [lo] and S. littoralis [ll].The activity ratio between ecdysone oxidase and 3a-reductase(s) indicated that the postulated sequence of the reactions (Fig. 1) is correct. The coexistence of a slow ecdysone oxidase and one or more fast 3-oxoecdysteroid 3a-reductases assures the functioning of the two-step reaction, but it is not compatible with the assumption of ecdysone oxidation as a side reaction unrelated to 3-epimerization [10,11,21]. This conchsion is supported by the good agreement between the K, reported here for the ecdysone oxidase (13.3 ? 1.8 pM) and the K, reported previously for the whole epimerization reaction (17.0 2 1.4 pM) [4]. A direct conversion of ecdysone to 3-epiecdysone without the intermediacy of 3-dehydroecdysone, although not firmly ruled out by these data, is much less likely. For P. brussicue [lo] and S. littoralis [ll],the two-step reaction sequence has been confirmed by isotope dilution experiments. Thus, the two-step conversion of 3p-hydroxyecdysteroids to 3a-hydroxyecdysteroids seems to be an inactivation mechanism common to lepidopteran larvae. The enzyme from C. vicina is the only other ecdysone oxidase for which kinetic data are available, and its K, for ecdysone is substantially higher Ecdysone Oxidase and Reductases 21 7 (42 and 98 WM, depending on the assay employed) [7,8] than that of the M. sextu enzyme. Two types of enzymes related to the 3-oxoecdysteroid 3-reductases are found in vertebrate tissues, 3a-hydroxysteroid dehydrogenases and 3P-hydroxysteroid dehydrogenases. The cytosolic 3a-hydroxysteroid dehydrogenases are assumed to convert potent steroid hormones to less potent metabolites, e.g. by conversion of 5wdihydrotestosterone to 3a-androstanediol[22]. The 36-hydroxysteroid dehydrogenases are found in microsomes and mitochondria and are involved in steroid hormone biosynthesis, e. g. by conversion of 3p-hydroxypregn-5-en- 20-one to progesterone [23]. An equivalent to the cytosolic 3P-hydroxysteroid dehydrogenase(s) of insects is not known in vertebrates, and membrane- bound hydroxysteroid dehydrogenases have not been explored in insects, but if present, they could add another level of complexity to the mechanism of 3-epimerization of ecdysteroids.

LITERATURE CITED

1. Lafont R, Koolman J: Ecdysone metabolism. In: Biosynthesis, Metabolism and Mode of Action of Invertebrate Hormones. Hoffmann J, Porchet M, eds. Springer-Verlag, Berlin, pp 196-226 (1984). 2. Thompson MJ, Weirich GF, Svoboda JA: Metabolism of insect molting hormones: Biocon- version and titer regulation. In: Morphogenetic Hormones of Arthropods. Gupta AP, ed. Rutgers University Press, New Brunswick, New Jersey, vol 1, pp 326-360 (1990). 3. Koolman J: Ecdysone metabolism. Insect Biochem 12,225 (1982). 4. Mayer RT, Durrant JL, Holman GM, Weirich GF, Svoboda JA: Ecdysone 3-epimerase from the midgut of Manduca sexta (L.). Steroids 34,555 (1979). 5. Nigg HN, Svoboda JA, Thompson MJ, Kaplanis JN, Dutky SR, Robbins WE: Ecdysone metabolism: Ecdysone dehydrogenase-isomerase. Lipids 9,971 (1974). 6. Koolman J: Ecdysone oxidase. Methods Enzymol111,419 (1985). 7. Koolman J, Karlson P: Ecdysone oxidase, an enzyme from the blowfly Calliphom erytlirocephala (Meigen). Hoppe-Seylers Z Physiol Chem 356, 1131 (1975). 8. Koolman J, Karlson P: Ecdysone oxidase: Reaction and specificity. Eur J Biochem 89, 453 (1978). 9. Somme-Martin G, Colardeau J, Lafont R: Conversion of ecdysone and 20-hydroxyecdysone into 3-dehydroecdysteroids is a major pathway in third instar Duosophih me/anogaster larvae. Insect Biochem 18,729 (1988). 10. Blais C, Lafont R: Ecdysteroid metabolism by soluble enzymes from an insect: Metabolic relationships between 3P-hydroxy-, 3a-hydroxy-, and 3-oxoecdysteroids. Hoppe-Seylers Z Physiol Chem 365,809 (1984). 11. Milner NP, Rees HH. Involvement of 3-dehydroecdysone in the 3-epimerization of ecdysone. Biochem J 231,369 (1985). 12. Weirich GF, Thompson MJ, Svoboda JA: In vitro ecdysteroid conjugation by enzymes of Manduca sexta midgut cytosol. Arch Insect Biochem Physiol3,109 (1986). 13. Thompson MJ, Weirich GF, Svoboda JA: Ecdysone 3-epimerase. Methods Enzymol111, 437 (1985). 14. Dinan L, Rees HH: Preparation of 3-epiecdysone and 3-epi-20-hydroxyecdysone. Steroids 32,629 (1978). 15. Spindler KD, Koolman J, Mosora F, Emmerich H: Catalytical oxidation of ecdysteroids to 3-dehydro products and their biological activities. J Insect PhysiolZ3, 441 (1977). 16. Thompson MJ, Kaplanis JN, Robbins WE, Dutky SR, Nigg HN: 3-Epi-20-hydroxyecdysone from meconium of the tobacco hornworm. Steroids 24,359 (1974). 17. Hoffmann JD, Lawson FR, Yamamoto R: Tobacco hornworms. In: Insect Colonization and Mass Production. Smith CN, ed. Academic Press, New York, pp 479-486 (1966). 218 Weirich et al.

18. Weirich GF, Adams JR Microsomal marker enzymes of Manduca sexta (L.) midgut. Arch Insect Biochem Physiol2,311 (1984). 19. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phe- nol reagent. J Biol Chem 193, 265 (1951). 20. Warren JT, Sakurai S, Rountree DB, Gilbert LI, Lee SS, Nakanishi K: Regulation of the ecdysteroid titer of Manduca sexta: Reappraisal of the role of the prothoracic glands. Pror Natl Acad Sci USA 85,958 (1988). 21. Weirich GF: Enzymes involved in ecdysone metabolism. In: Ecdysone. Koolman J, ed. Georg Thieme Verlag, Stuttgart, Federal Republic of Germany, pp 174-180 (1989). 22. Penning TM, Smithgall TE, Askonas LJ, Sharp RB: Rat liver 3a-hydroxysteroid dehydrogenase. Steroids 47, 221 (1987). 23. Chapman JC, Sauer LA: Intracellular localization and properties of 36-hydroxysteroid dehydrogenaseiisomerase in the adrenal cortex. J Biol Chem 254, 6624 (1979).