[28] Ecdysone 20-monooxygenase

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[28] E c d y s o n e 2 0 - M o n o o x y g e n a s e t By GUNTER F. WEIRICH

Hydroxylation of ecdysone2 to 20-hydroxyecdysone is generally considered to be the last step in the biosynthesis of the insect molting hormone. This conversion takes place in various tissues, e.g., fat body, Malpighian tubules, and midgut. 3 Depending on the species and tissue under study, ecdysone 20-monooxygenase can be found in mitochond r i a 4-6 and/or microsomes.7's In both of these cellular components, the enzyme is a cytochrome P-450 monooxygenase. This chapter describes procedures for the study of ecdysone 20-monooxygenases in midgut mitochondria and microsomes of the tobacco hornworm, Manduca sexta. [3H]Ecdysone Purification [23,24-3H]Ecdysone (New England Nuclear),9 to be used as substrate in the assay, is purified to >--95% radiopurity by reversed-phase highperformance liquid chromatography (RP-HPLC) on a radial compression Cls column (Waters Associates; 8 × 100 ram, 10-/.era particle size). [3H]Ecdysone is eluted isocratically with a mixture of methanol (Burdick & Jackson) and glass-distilled water (38/62) at 2 ml/min. After elution and collection of the [3H]ecdysone, the column is flushed with methanol/water (60/40) until effluent radioactivity is at background level. RP-HPLC EC 1.14.99.22. 2 2fl,3fl,14~,22R,25-Pentahydroxy-5fl-cholest-7-en-6-one. 3 M. J. Thompson, J. N. Kaplanis, G. F. Weirich, J. A. Svoboda, and W. E. Robbins, in "Regulation of Insect Development and Behaviour" (F. Sehnal, A. Zabza, J. J. Menn, and B. Cymborowski, eds.), p. 107. Wroclaw Technical University Press, Poland, 1981. 4 p. Johnson and H. H. Ress, Biochem. J. 168, 513 (1977). R. T. Mayer, J. A. Svoboda, and G. F. Weirich, Hoppe-Seyler's Z. Physiol. Chem. 359, 1247 (1978). 6 S. L. Smith, W. E. Bollenbacher, D. Y. Cooper, H. Schleyer, J. J. Wielgus, and L. I. Gilbert, Mol. Cell. Endocrinol. 15, 111 (1979). 7 R. Feyereisen and F. Durst, Eur. J. Biochem. 88, 37 (1978). s j. N. Kaplanis, G. F. Weirich, J. A. Svoboda, M. J. Thompson, and W. E. Robbins, in "Progress in Ecdysone Research" (J. A. Hoffmann, ed.), p. 163. Elsevier/North-Holland Biomedical Press, Amsterdam, 1980. 9 Mention of a company or proprietary product does not imply endorsement by the U.S. Department of Agriculture.

METHODS IN E N Z Y M O L O G Y , VOL. 1! 1

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in an acetonitrile/water system yielded unstable [3H]ecdysone solutions and was, therefore, deemed unsuitable for purification. Assay

Incubation The assay mixture contains 0.64 m M NADP +, 6.0 m M glucose 6phosphate, 1.0 unit glucose-6-phosphate dehydrogenase (from bakers' yeast, Sigma, Type XV), and mitochondria (2-I0 mg protein) or microsomes (2-6 mg protein) in 0.5 ml buffer solution (33 m M potassium phosphate, pH 7.8, 100 m M sucrose, 1 m M EDTA). To prevent losses of the mitochondrial enzyme activity, bovine serum albumin (Sigma, crystallized and lyophilized) is added to the assay mixture at 2.0 mg/ml. The reaction vessels are kept in a Dubnoff metabolic incubator at 30° and at 90-100 oscillations per min. After a 5-min preincubation, the reaction is started by the addition of 10 ~g [~H]ecdysone (4.6 Ci/mol) in 3-5 tA methanol. The reaction is stopped at an appropriate time by addition of 5.0 ml methanol. Protein is sedimented by a 15-rain centrifugation at 20,000 g, and the pellets are reextracted with another 5 ml methanol.

Isolation and Determination of 20-[3H]Hydroxyecdysone The methanol extract from each incubation mixture is dried under vacuum, redissolved in 1.0 ml methanol/water (1/9), and applied to a C~8 SEP-PAK cartridge (Waters). The cartridge is rinsed in sequence with 1.0 ml of methanol/water (1/9) and 5 ml methanol/water (3/7). Ecdysone and 20-hydroxyecdysone are eluted with 6 ml methanol/water (6/4; >90% recovery), the eluate is dried under vacuum, the residue dissolved in a small volume of methanol, and analyzed by RP-HPLC. Ecdysone and 20hydroxyecdysone are separated by isocratic elution with an acetonitrile (Burdick & Jackson)/water mixture from a t~Bondapak CI8 column (Waters Associates; acetonitrile/water, 20/80, 1 ml/min; retention times for ecdysone and 20-hydroxyecdysone, 19-22 and 8-10 rain, respectively). Alternately, a Radial Compression C~s column can be used (Waters Associates; 5 × 100 ram, 10-/~m particle size; acetonitrile/water, 18/82, 1 ml/min; retention times for ecdysone and 20-hydroxyecdysone, 14-15 and 5-6 min, respectively). The absorbance of the effluent is monitored at 254 nm. Some apolar tritium-labeled impurities of the samples are retained by the columns at the solvent strengths used for the separation. As these compounds tend to bleed slowly from the columns, they can cause errors in subsequent [3H]ecdysone or 20-[3H]hydroxyecdysone determinations.

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HOMOGENATE

I Accelerated to 3000 g [2.4 X 707 tad ~ sec-I ]

I

I Pellet rehomogenized in 3 vol buffer I Centrifuged as above

.

I Discard pellet

Supernatant

I Accelerated to 20,000 g [4.0 X 10 ~ tad ~ sec-~ ]

I

I

I

Pellet resuspended in 3 vol buffer

repeated two more times

Supernatant

I

I Accelerated to 13,000 g [2.0 X 108 tad 2 sec-l]

I MITOCHONDRIA

I

Centrifuged at 28,000 g [9,0 X 108 rad 2 see- I ]

f

I

Discard supernatant

Discard pellet

I

I

Supernatant

I Centrifuged at 80,000 g [3.6 X 10 ~° rad 2 sec-~]

I

I Pellet resuspended in 3 vol buffer

I POSTMICROSOMAL SUPERNATANT

I Centrifuged as above

I MICROSOMES

I

'1 Discard supernatant

FIG. 1. Isolation of mitochondria and microsomes. The centrifugations are described by the average g forces and the time integrals of the squared angular velocities [W = 981 × 60 (g min)/rav], given in brackets [H. Beaufay and A. Amar-Costesec, Meth. Membr. Biol. 6, 1 (1976)]. [Modified from G. F. Weirich and J. R. Adams, Arch. Insect Biochem. Physiol. 1, 311 (1984).1 A 5 - 1 0 rain flush o f the c o l u m n with acetonitrile/water (50/50) after each analysis is n e c e s s a r y to eliminate this problem. The c o n v e r s i o n rate o f e c d y s o n e to 2 0 - h y d r o x y e c d y s o n e is calculated from peak heights or peak areas o f the t w o c o m p o u n d s on U V tracings (after calibration o f the H P L C system), or from radioactivity determinations o f the corresponding effluent fractions. Comments

It is essential that assays o f the mitochondrial e n z y m e system are performed in a h y p o t o n i c incubation mixture. In isotonic buffer solutions

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(300 mM sucrose) mitochondria generally do not hydroxylate ecdysone, probably because intact mitochondrial membranes prevent its access to the enzyme system. 8 In assays of the mitochondrial ecdysone 20-monooxygenase, various hydrogen donors can be substituted for the NADPH-generating system (e.g., succinate, isocitrate, malate, N A D H ) ? All of these substrates are linked to the generation of NADPH by mitochondrial pathways. 5 These pathways, however, do not yield predictable N A D P H concentrations, and the substitute hydrogen donors are, therefore, generally not suitable for the determination of the monooxygenase activity. Mitochondria contain a pool of endogenous NADPH and/or other hydrogen donors that can sustain the ecdysone 20-hydroxylation without added cosubstrate(s). 8 The NADPH-generating system is added to optimize the N A D P H supply in the assay mixtures. Microsomes, on the other hand, do not have permeability barriers for ecdysone. They have no endogenous pool of NADPH and cannot utilize other hydrogen donors, io Isolation of Mitochondria and Microsomes Fifth-instar larvae of M. sexta are collected on the first day of "wandering ''11,12 and cooled in ice. The midguts are dissected and freed from fat body, Malpighian tubules, trachea, and gut contents under ice-cooled 10 mM Tris-HCl buffer, pH 7.4, containing 300 mM sucrose and 1 mM EDTA (isolation buffer). The cleaned and rinsed guts are then blotted dry on tissue, weighed, minced with scissors, and homogenized in 6 ml isolation buffer per gram tissue (6 "volumes") in a Ten Broeck homogenizer cooled in ice. The homogenizer is initially operated by hand until the tissue is broken up and suspended in the buffer. The homogenization is then completed by two strokes with the motor-driven pestle at approximately 650 rpm. The homogenate is fractionated as outlined in Fig. 1. In this scheme the duration of all centrifugations (except for the sedimentation of microsomes) is reduced to a minimum by increasing the speed, 13 and some centrifugations consist only of acceleration and deceleration, with no plateau phase in between. Mitochondrial and microsomal pellets are resuspended by gentle mixing with isolation buffer in glass-Teflon homogenizers. Isolation of mitochondria and microsomes from insect tissues can also be accomplished by centrifugation in a sucrose density gradient. 7 Mitoto G. F. H j. W. t~ H. F. i~ L. L.

Weirich, unpublished observation (1979). Truman and L. M. Riddiford, J. Exp. Biol. 60, 371 (1974). Nijhout and C. M. Williams, J. Exp. Biol. 61, 481 (1974). Keeley, Comp. Biochem. Physiol. B 46, 147 (1973).

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chondrial preparations contain a small proportion of microsomes even after repeated washings. T o assess the extent of this contamination, N A D P H - c y t o c h r o m e c reductase activity has to be determined for both the isolated mitochondria and microsomes.Z4 Properties F o r the mitochondrial e n z y m e system, the apparent Km and Vma~ for e c d y s o n e are 18.3 - 6 . 8 / x M and 46.6 -+ 14.2 pmol/min/mg protein, res p e c t i v e l y ) The microsomal system has not been characterized. 14G. F. Weirich and J. R. Adams, Arch. Insect Biochem. Physiol. 1, 311 (1984).

[29] Measurement and Characterization of Ecdysteroid Receptors B y BECKY A. SAGE and JOHN D. O'CONNOR

Introduction The modulation o f cellular responses to steroid hormones by intracellular steroid r e c e p t o r molecules has been the central component o f the interpretive paradigm of steroid action since the early 1960s. ~-3 H o w e v e r , it was not until 1978 that the unequivocal demonstration of ecdysteroid receptors in cells and tissues o f D r o s o p h i l a m e l a n o g a s t e r was forthcoming. 4,5 During the intervening years between the first demonstration of intracellular steroid receptors and the present time there have been four characteristics which have emerged as the sine qua non of receptors in general and a fifth characteristic particular to systems responding to steroid hormones. The first four characteristics of a receptor molecule are saturability, specificity, high affinity for the biological ligand, and the association o f the biological response with the presence of the receptor. Paraphrasing this last point: in the absence o f a high affinity, highly spel E. V. Jensen and H. I. Jacobson, Recent Prog. Horm. Res. 18, 307 (1962). 2 W. D. Noteboom and J. Gorski, Arch. Biochem. Biophys. 111, 559 (1965). 3R. J. B. King, J. Gordon, and D. R. lnman, Endocrinology 32, 9 (1965). 4 p. Maroy, R. Dennis, C. Beckers, B. A. Sage, and J. D. O'Connor, Proc. Natl. Acad. Sci. U.S.A. 759 6035 (1978). 5 M. A. Yund, D. S. King, and J. W. Fristrom, Proc. Natl. Acad. Sci. U.S.A. 75, 6039 (1978). METHODS IN ENZYMOLOGY, VOL. 111

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