Characterization of ATP: Ecdysone 3-acetate 2-phosphotransferase (ecdysone 3-acetate 2-kinase) in Schistocerca gregaria larvae

Insect Bioehem. Molec. Biol. Vol. 23, No. 1, pp. 73-79, 1993

0965-1748/93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Ltd

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Characterization of ATP:Ecdysone 3-Acetate 2-Phosphotransferase (Ecdysone 3-Acetate 2-Kinase) in Schistocerca gregaria Larvae MOHAMED KABBOUH,* HUW H. REES*t

Soluble ATP:ecdysone 3-acetate 2-phosphotransferase from fat body of day 7 last instar larvae of Schistocerca gregaria was characterized. The phosphotransferase activity was present in all tissues examined, but the highest activity was obtained in fat body eytosol. A single peak of ATP:ecdysone 3-acetate 2-phosphotransferase activity was obtained by DEAE-cellulose chromatography. The enzyme has a molecular weight of ca 45 kDa, determined by gel filtration on Sephadex G-150. Ecdysone 3-acetate was the preferred substrate for the enzyme, whereas ecdysone 2-acetate and ecdysone were not significantly phosphorylated. The phosphotransferase activity survived freezing at --20°C but was totally abolished by heat at 60°C for 10 min. The reaction rate was linear for 60 rain and with increasing protein concentration up to 2mg[ml. Optimal pH was about 7.5. The phosphotransferase activity required the presence of ATP, the apparent Km for ATP being 91.1 ~M. The enzyme exhibited an apparent Km for ecdysone 3-acetate of 10.5 pM and a maximal specific activity of 91.1 pmol[min/mg protein. Ecdysone and 20-hydroxyecdysone did not inhibit the phosphotransferase activity. Maximal enzyme activity was obtained in the presence of 5-10 mM Mg 2+ while Ca ~+ was inhibitory with an ICs0 value of 1 mM. Investigation of the variation in activity of the phosphotransferase during development of the last instar larvae showed that the phosphotransferase activity increased significantly during the second half of the instar when the titre of endogenous ecdysteroids is at its highest level. Phosphorylation Ecdysone3-acetate Ecdysteroids Sehistocerca gregaria acetate 2-phosphotransferase Ecdysone3-acetate 2-kinase

INTRODUCTION

Fat body ATP:ecdysone 3-

many ecdysteroid phosphates have been identified in 3-Acetylecdysteroid 2-phosphates have been identified as different stages of development of several insect species, only one phosphotransferase, involved in formation of inactivation products of ecdysteroids in developing eggs as well as in postembryonic life stages of several insect ecdysteroid 22-phosphates in follicle cells of S. gregaria, species (for review see Rees, 1989). Two different en- has been characterized to date (Kabbouh and Rees, zymic systems are involved in their formation. Firstly, it 1991b). Weirich et al. (1986) reported the formation of has been shown recently in Schistoeerea gregaria larvae a variety of phosphoesters of ecdysteroids (not identthat ecdysone (or 20-hydroxyecdysone) is converted into ified) by Manduca sexta midgut cytosol in the presence of ATP and Mg 2÷, suggesting the involvement of several its corresponding 3-acetate derivative by a microsomal ATP:ecdysteroid phosphotransferases in ecdysteroid acetyl-CoA:ecdysone 3-acetyltransferase (Kabbouh and metabolism. In locusts, the phosphorylation at C-22 in Rees, 1991a). Evidence from in vivo studies suggest that follicle cells of mature females is involved in formation ecdysone 3-acetate (or 20-hydroxyecdysone 3-acetate) of ecdysteroid phosphate esters. These are believed to be serves as substrate for a phosphotransferase yielding inactive, storage forms of hormone which are passed 3-acetylecdysone 2-phosphate (or 3-acetyl-20-hydroxyinto the eggs and, apparently, after hydrolysis provide a ecdysone 2-phosphate; Modde et al., 1984; Gibson et al., source of ecdysteroid in the early stages of embryonic 1984). The combination of C-3 acetylation and C-2 phosphorylation represent the major inactivation reac- development (for review, see Isaac and Rees, 1984; tions in locusts, since 3-acetylphosphate derivatives rep- Hoffmann and Lagueux, 1985). In contrast, phosphorylresent the major conjugates excreted in faeces. While ation at C-2 is presumed to be involved in formation of hormone inactivation products which are found in faeces of larvae (Modde et al., 1984; Gibson et al., 1984) and developing eggs (Isaac and Rees, 1984). *Department of Biochemistry,Universityof Liverpool,P.O. Box 147, We now report characterization of the enzymic system Liverpool L69 3BX, England. ~Author for correspondence. responsible for the phosphorylation at C-2 of ecdysone 73

74

MOHAMED KABBOUHand HUW H. REES

3-acetate in fat body cytosol of day 7, fifth instar S. gregaria, a time in development when the endogenous ecdysteroid titre is low under the current rearing conditions (Gande et al., 1979). Ecdysone 3-acetate, the substrate, could be obtained by chemical acetylation of ecdysone (Galbraith and Horn, 1969) but the yield is very low and this procedure could not produce sufficient ecdysone 3-acetate required for biochemical study of the phosphotransferase. However, this problem was overcome after characterization of the acetyltransferase involved in its biological formation. Using microsomal fraction from gastric caecae of day 7, fifth instar larvae, having a specific activity of 7.2 nmol/min/mg protein the acetyl-CoA: ecdysone 3-acetyltransferase, produced in the presence of acetyl-CoA, exclusively high amounts of ecdysone 3-acetate. This was quickly purified by HPLC and stored dry to prevent chemical migration of the acetate to position C-2 (Kabbouh and Rees, 1991a). MATERIALS AND METHODS

[_nsects Locusts were reared in the gregarious phase with a 12 h light period at a daytime temperature of 37°C, while at night the temperature dropped to 25°C. The insects were fed on a diet of fresh cabbage and wheat bran. Under these conditions, the fifth (last) larval instar lasted ca 8 days. Chemicals [23,24-3H2]ecdysone (89Ci/mmol) was from New England Nuclear, Boston, Mass. Unlabelled ecdysone was from Simes (Milan, Italy). Tritiated ecdysone 3acetate and unlabelled ecdysone 3-acetate were prepared from ecdysone using the same conditions as for the acetyl-CoA: ecdysone 3-acetyltransferase assay (Kabbouh and Rees, 1991a) and purified by reversedphase HPLC. DEAE-cellulose (DE 52) was from Whatman. Sephadex G-150 was purchased from Pharmacia.

DEAE-cellulose chromatography Fat body from 200 fifth instar larvae (day 7) was homogenized in a Teflon-glass Potter-Elvehjem homogenizer in 20 ml of 5 mM Tris-HC1 buffer, pH 7.5, containing 2mM KF and 2raM EDTA (buffer B). Following centrifugation at 150,000 g for 60 min, 20 ml of the resulting supernatant was loaded on a DEAE-cellulose column (0.9 x 22 cm; Whatman DE52) which had been pre-equilibrated with buffer B. The column was washed with 30 ml buffer B prior to the initiation of a 60 ml linear salt gradient (0-400 mM NaC1 in the same buffer) at a flow rate of ca 12 ml/h. 2 ml fractions were collected and 50/~1 of each fraction was assayed for phosphotransferase activity under standard assay conditions (see later). The fractions containing phosphotransferase activity were desalted using Sephadex G-25M (PD-10 columns, Pharmacia) and 87.5 #I from each desalted fraction assayed for phosphotransferase activity. These fractions were pooled and served as a source of enzyme in biochemical characterization of the phosphotransferase as well as in estimation of its molecular weight by gel filtration. Gel filtration chromatography 1.5ml (2rag protein) of the pooled fractions after DEAE-cellulose chromatography were loaded on a Sephadex G-150 column pre-equilibrated and eluted with 50mM K-phosphate buffer, pH 7.5, containing 0.1 M NaC1 and 0.6% sodium azide (buffer C). Flow rate was 3 ml/h and 1 ml fractions were collected. Fractions were assayed for phosphotransferase activity under the standard assay conditions (see next). The column was calibrated with protein standards having the following molecular weights: pyruvate kinase (240,000); lactate dehydrogenase (140,000); albumin (67,000); ovalbumin (45,000); carbonic anhydrase (30,000); trypsin inhibitor (21,000) and cytochrome c (12,400). Fractions were assayed for protein by the method of Lowry et al. (1951).

Phosphotransferase assay All assays were carried out in duplicate and the results were expressed as means of these determinations. For Km Phosphotransferase activity in different tissues Fat body, Malpighian tubules, gut and gastric caecae determination, data were processed by computer using were excised from 10 fifth instar larvae (day 7) in 0.l M the Enzpack 3 program, version 3.0, written by Peter A. sodium phosphate buffer, pH 7.4 containing 50 mM Williams and Bogus N. Zaba (School of Biological KC1, 2raM 2-mercaptoethanol, 0.1% BSA, 1 mM Sciences, University College of North Wales, Bangor, EDTA and 0.25 M sucrose (buffer A), rinsed several Wales) and published by Biosoft, 22 Hills Road, Camtimes and homogenized in a Teflon glass Potte~ bridge, England, 1989. The standard assay for ATP:ecdysone 3-acetate 2Elvehjem homogenizer in the same buffer solution (4 ml). The homogenate was centrifuged immediately phosphotransferase activity contained the following in at 150,000g for 60min and the supernatant (cytosol) buffer A: ecdysone 3-acetate (0.25#Ci, 10-100#M), used as the source of enzyme. All operations were carried ATP (2mM)/Mg 2+ (10raM) and cytosol (25-50#1, out at 0~°C. The phosphotransferase activity was 200~400 #g protein) or pooled column fractions determined under the standard assay conditions using (50-100/~1, 100-200#g protein) in a total volume of 30 #1 of cytosol for fat body and Malpighian tubules 200 #1. After incubation at 37°C for 30 rain, the reaction and 20 #1 of cytosol for gastric caecae and gut (see was stopped by addition of methanol (200/~1). The mixture was then centrifuged for 5 rain at full speed later).

ECDYSONE 3-ACETATE 2-KINASE

(8800g) in an MSE Microcentaur centrifuge and an aliquot (50-100 #1) was analyzed directly by HPLC.

High-performance liquid chromatography HPLC separations were carried out on a reversedphase column (radial compression module Nova-Pak C~8 column, 10 cm x 8 ram, particle size 4 #m, Waters Assoc.) eluted at 1 ml/min with a linear gradient (30 min) of acetonitrile in Tris-HC104 (20 mM, pH 7.5) changing from 1:9 (v/v) to 4:6 (v/v). The radioactivity was monitored by an on-line radioactivity monitor (A-200 Flo-one/Beta, Radiomatic Instruments, Canberra Company, U.S.A.). Mass spectrometry Negative-ion fast atom bombardment (FAB) mass spectrometry was carried out using a primary atom beam of xenon with energy 8 KeV. The sample, as solution in methanol, was added to glycerol on the probe tip prior to FAB.

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Identification of reaction product in the standard phosphotransferase assay Cytosol from fat body was incubated with [3H]ecdysone 3-acetate under the standard phosphotransferase assay conditions and the product, after removal of protein, analysed in the methanolic solution by HPLC. Since most phosphotransferases are cytosolic, the possible occurrence of such activity in other subcellular fractions was not investigated. Figure l(a) gives the reversed-phase HPLC radiochromatogram of the product obtained after incubation of fat body cytosol with tritiated ecdysone 3-acetate, 20 #M unlabelled ecdysone 3-acetate and ATP (2mM)/Mg 2+ (10mM). Only one radioactive product was obtained corresponding to the position of elution of authentic 3-acetylecdysone 2-phosphate. This radioactive peak was collected and treated at room temperature with a 0.6% solution of K z C O 3 in methanol:water (9: 1, v/v) for 2hr. Figure l(b) gives the reversed-phase HPLC radiochromatogram of the product obtained after such treatment, which was shown to be ecdysone 2phosphate, accompanied by unreacted 3-acetylecdysone 2-phosphate, by co-chromatography on HPLC with authentic material. The molecular weight of the 3-acetylecdysone 2-phosphate product was confirmed by negative ion FAB mass spectrometry. For this, ecdysone 3-acetate (100 #g) was incubated in 1 ml buffer A in the presence of fat body cytosol (2 mg protein) and ATP (2 mM)/Mg 2+ (10 mM). 3-Acetylecdysone 2-phosphate was purified by HPLC

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Protein determination Protein concentrations were determined by the procedure of Lowry et al. (1951) with bovine serum albumin (BSA, Sigma) as standard.

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FIGURE 1. Reversed-phase HPLC radiochromatograms of (a) the reaction product formed during incubation of fat body cytosol with ecdysone 3-acetate (0.25/~Ci, 20#M) and ATP (2mM)/Mg 2+ (10mM) and (b) the reaction product after treatment by K2CO 3. Chromatography was on a Nova-Pak C~8 column (10cm x 8mm, particle size 4 # m) eluted with a linear gradient (30 min) of acetronitrile in Tris-HC104 (20 mM, pH 7.5) changing from 1:9 (v/v) to 2:3 (v/v). The positions of elution of authentic ecdysone 2-phosphate (E2P), 3-acetylecdysone 2-phosphate (E3A2P), and ecdysone 3-acetate (E3A) are shown.

and analysed by negative ion FAB mass spectrometry. The spectrum showed a distinct [ M H ] - ion at m/z 585 which indicated that the molecular weight of the enzymic reaction product (3-acetylecdysone 2-phosphate) was 586. Furthermore, one typical fragment at m/z 467 [(M-H) --(C22-C27)] confirmed that the phosphate moiety is linked to the nucleus. Taken together with cochromatography of the substance with the authentic compound, the results confirm identity of the reaction product as 3-acetylecdysone 2-phosphate.

Phosphotransferase activity in different tissues Cytosols from fat body, Malpighian tubules, gut and gastric caecae were separately incubated, under the standard assay conditions, with ecdysone 3-acetate and ATP/Mg 2÷. Figure 2 gives the phosphotransferase activities obtained in these various tissues. It is apparent

76

MOHAMED KABBOUH and HUW H. REES 150

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FIGURE 2. Comparison of specific activities of phosphotransferase in different tissue cytosols. Cytosols were prepared from fat body (FB), Malpighian tubules (MT), gastric caecae (GC), and gut and the phosphotransferase activity was assayed under the standard conditions (see Materials and Methods).

from this figure that ATP:ecdysone 3-acetate 2-phosphotransferase activity was present in all tissues examined but the highest phosphotransferase specific activity was obtained in fat body cytosol and to a lesser extent in Malpighian tubule cytosol. In both gut and gastric caecae the specific activity of phosphotransferase was low but significant (7.5 and 8.5%, respectively, of the activity obtained in fat body). The relatively high degree of phosphorylation by fat body led to the use of this tissue in investigations on the biochemical characteristics of the phosphotransferase as well as its variation in activity during development of fifth instar larvae.

FIGURE 3. DEAE-cellulose (DE-52) chromatography of solubie ATP: ecdysone 3-acetate 2-phosphotransferase (150,000 g supernatant) from larval fat body. The phosphotransferase activity was measured under the standard assay conditions (see Materials and Methods).

retained after freezing at - 2 0 ° C for over 1 month. The reaction rate was followed for 60 min and was found to be linear during that period [Fig. 5(a)] and with protein concentration up to 2 mg/ml [Fig. 5(b)]. Optimal pH was about 7.5 [Fig. 5(c)]. In the absence of added exogenous ATP no detectable phosphotransferase activity was obtained. The apparent Km for ATP was 91.1 # M [Fig. 5(d)]. The Km of the phosphotransferase for ecdysone 3acetate was determined for the cytosol after chromatography on DEAE--cellulose. As shown in Fig. 6 the value of the apparent Km was 10.5 p M and Vmaxwas 91.1 pmol/min/mg protein. Ecdysone and 20-hydroxyecdysone did not inhibit the phosphotransferase activity (data not shown). When the specificity of the phosphotransferase for ecdysone acetates was investigated

DEAE-cellulose chromatography and molecular weight estimation Anion exchange chromatography of the 150,000g supernatant from larval fat body yielded a single peak of ATP:ecdysone 3-acetate 2-phosphotransferase which eluted between 75-170 mM NaC1 (Fig. 3). Fractions of this peak were desalted on Sephadex G-25M columns (PD-10, Pharmacia) using buffer B and the phosphotransferase activity was determined in each desalted fraction. The results (Fig. 3) show that sodium chloride inhibited the phosphotransferase activity (about 40% inhibition at the summit of the peak). Recovery of ATP:ecdysone 2-phosphotransferase activity from the DEAE~ellulose column was >90%. The fractions of the peak were pooled and an aliquot loaded on a Sephadex G-150 column. The ATP : ecdysone 3-acetate 2-phosphotransferase activity was eluted as a single peak at apparent Mr = 45,000 (Fig. 4). Biochemical characteristics of the ATP:ecdysone 3acetate 2-phosphotransferase The enzymic activity was totally abolished by heating the cytosol at 60°C for 10min, but the activity was

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FIGURE 4. Determination of molecular weight of soluble ATP:eedysone 3-acetate 2-phosphotransferase using Sephadex G-! 50 gel filtration. The phosphotransferase activity was measured under the standard assay conditions (see Materials and Methods). Volume of elution of standard proteins is plotted against log MW: [, pyruvate kinase; 2, lactate dehydrogenase; 3, albumin; 4, ovalbumin; 5, carbonic anhydrase; 6, trypsin inhibitor; 7, cytochrome c.

ECDYSONE 3-ACETATE 2-KINASE (b)

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FIGURE 5. Effects of incubation time, protein concentration, pH and ATP on soluble ATP:ecdysone 3-acetate 2-phosphotransferase activity. The reaction mixture contained ATP (2 mM)/Mg 2+ (10 mM) and the phosphotransferase activity was measured under the standard assay conditions as described in Materials and Methods except for the parameter under study. (a) Incubation time varied from 2 to 60 min; (b) protein concentration varied from 0.1 to 3.75 mg/ml; (c) constant ionic strength sodium phosphate buffer was used over the pH range 5.5-8.5; and (d) ATP concentration varied from 0.05 to 5 mM, Km(ATP) = 91.1/~M; standard error, SE(Km)= 6.99. using e c d y s o n e 3-acetate a n d ecdysone 2-acetate, only e c d y s o n e 3-acetate was c o n v e r t e d into 3-acetylecdysone 2 - p h o s p h a t e , suggesting t h a t the p h o s p h o r y l a t i o n o f e c d y s o n e acetate o c c u r r e d specifically on p o s i t i o n 24 ,

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C-2. E c d y s o n e was p o o r l y c o n v e r t e d into e c d y s o n e 2-phosphate. T h e p h o s p h o t r a n s f e r a s e activity was m a r k e d l y influenced b y M g 2+ with a m a x i m a l l y effective c o n c e n t r a t i o n o f 1 0 m M (Fig. 7). H o w e v e r , a strong i n h i b i t i o n was o b t a i n e d with C a 2 ÷ in the presence o f s a t u r a t i n g M g 2÷ ( 1 0 m M ) , with an ICs0 o f 1 m M (50% i n h i b i t i o n was o b t a i n e d with a c o n c e n t r a t i o n o f 1 m M C a 2÷) a n d nearly c o m p l e t e i n h i b i t i o n at 100 m M (Fig. 7).

Variation in activity of the ATP:ecdysone 3-acetate 2-phosphotransferase during development of last instar larvae

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FIGURE 6. Lineweave~Burk plot of the soluble ATP:ecdysone 3-acetate 2-phosphotransferase activity. Assay conditions were described in Materials and Methods except that the concentration of unlabelled ecdysone 3-acetate varied from 10 to 200 #M. The reaction mixture was supplied with ATP (2 mM)/Mg2+ (10 mM) as co-factors. Km = 10.5 #M; standard error, SE(Km)= 0.595.

C y t o s o l s were p r e p a r e d f r o m fat b o d y (10 insects) at different stages o f last instar d e v e l o p m e n t between d a y 0 ( m o u l t i n g into last i n s t a r larvae) a n d d~,, 8 ( a d u l t moult). T h e p h o s p h o t r a n s f e r a s e activity w~ aeasured u n d e r the s t a n d a r d assay c o n d i t i o n s . T h e results (Fig. 8) s h o w t h a t the p h o s p h o t r a n s f e r a s e activity, expressed per m g protein, was relatively high at the time o f m o u l t i n g into last i n s t a r larvae (day 0) a n d u n d e r w e n t little v a r i a t i o n d u r i n g the first 4 d a y s o f the instar. T h e n the activity increased significantly in the last 4 d a y s o f the instar, finishing > 2-fold the activity at m i d instar a n d > 3-fold the activity at d a y 0. A l t h o u g h the activity,

78

M O H A M E D K A B B O U H and H U W H. REES

enzyme and that this phosphorylation is very specific. The reaction product, 3-acetylecdysone 2-phosphate, was identified by a combination of reversed-phase HPLC, its partial hydrolysis with K 2 C O 3 , and negativeFAB mass spectrometry. The release of ecdysone 2phosphate after treatment of the reaction product with K2CO3 solution and the presence of a fragment at rn/z 467 [(M-H)--(C22-C27)] in the mass spectrum confirmed that the phosphorylation of ecdysone 3-acetate occurred exclusively on the C-2 position. The phosphotransferase has a wide distribution since all tissues examined contained some activity. However, it is of significance to note that the bulk of the phosphotransferase activity (> 90%) was found in two physiologically different tissues--on the one hand, in fat body which is involved in ecdysteroid metabolism and on the other hand in Malpighian tubules, which are responsible mainly for ecdysteroid excretion in addition to hormone metabolism. The enzyme preparations from gut and gastric caecae (site of absorption of nutrients) had very low specific activities, although these tissues contain the bulk of the acetyl-CoA:ecdysone 3-acetyltransferase (>95%, unpubl, results) which is the generator of the substrate for the phosphotransferase, ecdysone 3-acetate. In fat body, the specific activity of the phosphotransferase (127 pmol/min/mg protein) represents double that of the acetyltransferase (65pmol/min/mg protein), suggesting that some of the substrate, viz. ecdysone 3-acetate, may be generated outside the fat body. Since the acetylation reaction occurred mainly in digestive tissues (gastric caecae and gut), in vivo the substrate ecdysteroid could conceivably be of endogenous or dietary origin. Taken together, the tissue distribution of the acetyltransferase and the phosphotransferase suggest that these two enzymic systems play an efficient role in inactivation of endogenous hormones as well as exogenous phytoecdysteroids with the phosphotransferase using the reaction product of the acetyltransferase as substrate regardless of its origin.

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F I G U R E 7. Effect of Mg 2+ ( 1 ) and Ca 2+ ( 0 ) on soluble ATP:ecdysone 3-acetate 2-phosphotransferase activity. Assays were performed under standard conditions with A T P concentration being maintained at a constant 2 mM. Initial activity was measured in the presence of 10 m M MgC12. Ca 2+ was added as CaC12.

expressed per insect equivalent, showed a somewhat similar trend, it is important to note that the phosphotransferase activity per insect equivalent was very low at day 0 and increased sharply from day 2 to a peak at days 5-6. In both cases, high activity is observed after day 4. DISCUSSION Ecdysone 3-acetate, the reaction product of acetylCoA:ecdysone 3-acetyltransferase (Kabbouh and Rees, 1991a), was phosphorylated at C-2 by a soluble ATP:ecdysone 3-acetate 2-phosphotransferase which was characterized in fat body cytosol of day 7, fifth instar larvae of S. gregaria. Ecdysone 2-acetate and ecdysone were not phosphorylated by this phosphotransferase under the same standard assay conditions, suggesting that the acetate at the C-3 position plays a major role in the recognition of the substrate by the

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Larval development (days)

F I G U R E 8. Variation of the ATP:ecdysone 3-acetate 2-phosphotransferase activity during larval development. Fat body cytosol was prepared every day of development and the phosphotransferase activity was measured under the standard assay conditions (see Materials and Methods).

ECDYSONE 3-ACETATE 2-KINASE D E A E - c e l l u l o s e c h r o m a t o g r a p h y o f fat b o d y c y t o s o l revealed only a single p e a k o f A T P : e c d y s o n e 3-acetate 2 - p h o s p h o t r a n s f e r a s e activity which gives a m o l e c u l a r weight o f ca 45 k D a ( m o n o m e r ) b y gel filtration on S e p h a d e x G-150. It is very difficult to relate this p r o t e i n to o t h e r k n o w n p h o s p h o t r a n s f e r a s e s a n d no d a t a exist c o n c e r n i n g this enzymic system in o t h e r insect species. The A T P : e c d y s o n e 3-acetate 2 - p h o s p h o t r a n s f e r a s e has a cytosolic localization, exhibits classical M i c h a e l i s - M e n t e n kinetics a n d has an a p p a r e n t Km for e c d y s o n e 3-acetate o f 1 0 . 5 # M . T h e enzyme requires b o t h A T P (Kin = 91.1 ~ M ) a n d M g 2+ ( m a x i m a l activity in the presence o f 1 0 r a M ) for activity. The p h o s p h o transferase activity was s t r o n g l y inhibited in the presence o f C a 2+. T a k e n together, these results suggest t h a t A T P : e c d y s o n e 3-acetate 2 - p h o s p h o t r a n s f e r a s e somew h a t resembles the A T P : 2 - d e o x y e c d y s o n e 2 2 - p h o s p h o transferase f r o m follicle cells o f vitellogenic females, a l t h o u g h the p h o s p h o t r a n s f e r a s e f r o m fat b o d y has lower affinity for the ecdysteroid, e c d y s o n e 3-acetate, a n d higher affinity for A T P t h a n the one f r o m follicle cells. E x a m i n a t i o n o f the v a r i a t i o n in activity o f A T P : e c d y s o n e 3-acetate 2 - p h o s p h o t r a n s f e r a s e d u r i n g last i n s t a r d e v e l o p m e n t s h o w e d t h a t when the activity was expressed either as per m g p r o t e i n o r p e r insect equivalent, there was a significant i n c r e m e n t in the second h a l f o f the instar. C o m p a r i s o n o f this enzyme titre with t h a t o f the e n d o g e n o u s ecdysteroids shows t h a t the t o t a l enzyme titre is increasing several d a y s before the rise in e c d y s t e r o i d c o n c e n t r a t i o n . Therefore, it is likely t h a t the enzyme is n o t only involved in i n a c t i v a t i o n o f e n d o g e n o u s h o r m o n e s at the time o f high e n d o g e n o u s e c d y s t e r o i d titre, b u t a role in i n a c t i v a t i o n o f e x o g e n o u s p h y t o e c d y s t e r o i d s is possible since the enzymic activity is relatively high in the first h a l f o f the instar where the

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e n d o g e n o u s e c d y s t e r o i d titre is negligible ( G a n d e et al., 1979). REFERENCES

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financial support, Mr Mark C. Prescott for FAB mass spectrometric analysis and Mrs Army Dock-Kabbouh for secretarial assistance.