Purification, kinetic characterization, and molecular cloning of a novel enzyme ecdysteroid-phosphate phosphatase.

From eggs of the silkworm Bombyx mori, we isolated a novel enzyme that is involved in the conversion of physiologically inactive conjugated ecdysteroids, such as ecdysone 22-phosphate and 20-hydroxyecdysone 22-phosphate, to active free ecdysteroids. This enzyme, called ecdysteroid-phosphate phosphatase (EPPase), was located in the cytosol fraction and differed from nonspecific lysosomal acid phosphatases in various enzymic properties. EPPase was purified about 3,000-fold to homogeneity by seven steps of column chromatography. The cDNA clone encoding EPPase was isolated by reverse transcription polymerase chain reaction using degenerate primers on the basis of the partial amino acid sequence obtained from purified EPPase and by subsequent 3'- and 5'-rapid amplification of cDNA ends. The full-length cDNA of EPPase was found to be composed of 1620 bp with an open reading frame encoding a protein of 331 amino acid residues. A data base search showed that there was no functional protein with the amino acid sequence identical to that of EPPase. Northern blot analysis revealed that EPPase mRNA was expressed predominantly during gastrulation and organogenesis in nondiapause eggs but was not detected in diapause eggs whose development was arrested at the late gastrula stage. In nondiapause eggs, the developmental changes in the expression pattern of EPPase mRNA corresponded closely to changes in the enzyme activity and in the amounts of free ecdysteroids in eggs.

In B. mori, recently, it has been demonstrated that the continuous supply of 20-hydroxyecdysone (20E), 1 which has been demonstrated to be an active molecule in B. mori eggs (13), is required for embryonic development and that a deficiency of 20E induces embryonic diapause (13); that is, the cessation of embryonic development at the late gastrula stage (14,15). Furthermore, 20E was demonstrated to be formed by de novo synthesis (16 -18) and dephosphorylation of 20-hydroxyecdysone 22-phosphate (20E22P) and ecdysone 22-phosphate (E22P) (in the case of the latter, dephosphorylation was followed by hydroxylation at C-20 position) (18).
Although the potential role of ecdysteroid-phosphates as a "reservoir" of active ecdysteroids has been suggested in various insect eggs as mentioned above, little or no attention has been paid to the enzyme catalyzing the dephosphorylation, ecdysteroid-phosphate phosphohydrolase (ecdysteroid-phosphate phosphatase (EPPase)) ( Fig. 1). We now report the purification, characterization, and cDNA cloning of EPPase from B. mori eggs. This is the first report of a novel phosphatase that specifically hydrolyzes ecdysteroid-phosphates.

EXPERIMENTAL PROCEDURES
Animals-Moths of the silkworm B. mori that had been destined to produce diapause eggs, the commercially available bivoltine race (kinsyuϫshowa), were used as the source of eggs. In diapause eggs, embryos ceased development at the late gastrula stage (48 -72 h after oviposition) (14,15). The diapause nature of the eggs was artificially changed to a nondiapause nature by the following treatments: (i) To avoid diapause initiation, prospective diapause eggs were soaked in HCl solution (specific gravity, 1.11 at 15°C) 20 h after oviposition (15). (ii) To obtain female moths destined to produce nondiapause eggs, the subesophageal ganglion, the source of diapause hormone, was removed soon after larval-pupal ecdysis (15). In both cases, the nondiapause eggs obtained continued to develop and larvae hatched 10 days after oviposition. For enzyme purification, HCl-treated 3-day-old nondiapause eggs were used. For RNA extraction, nondiapause eggs laid by the moths with the subesophageal ganglion removed were used, because ommochromes, which disturb first strand cDNA synthesis, are not synthesized in the nondiapause eggs.
Enzyme Assay-The EPPase activity was measured using E22P as the substrate. E22P was synthesized chemically (19). The amount of ecdysone (E) converted from E22P was quantified by radioimmunoassay (RIA) using antiserum N-6 (provided by Dr. A. Mizoguchi, Nagoya University) generated against 20E-6-carboxy methyloxime hapten. This antiserum exhibited over 4,000-fold stronger reactivity against E than against E22P (19). Therefore, the amount of E in the reaction mixture was quantified by RIA without isolating E from E22P. RIA was conducted as described previously (20). For the measurement of EPPase activity, the reaction mixture was made up to a final volume of 100 l with 200 mM Tris-HCl buffer, pH 7.5, containing 0.1% bovine serum albumin, 100 nM E22P, and 50 l of the enzyme solution. The reaction was initiated by adding the enzyme solution, and the reaction mixture was incubated at 37°C for 30 min. The reaction was stopped by the addition of methanol, and the mixture was centrifuged at 3,500 rpm for 30 min to precipitate the protein. Aliquots of the supernatant were dried using a centrifugal evaporator. The residue was dissolved in 50 mM borate buffer, pH 8.4, for use in RIA.
Acid phosphatase activity was measured using p-nitrophenylphosphate (pNPP), which is generally used as the substrate of phosphatase (21)(22)(23)(24). The assay mixture containing 10 mM pNPP and 200 mM acetate buffer, pH 4.0, was incubated with 50 l of the enzyme solution at 37°C for 15 min. After the reaction was stopped by the addition of 0.1 M NaOH, the absorbance of p-nitrophenol (pNP) formed was measured at 405 nm.
Protein Assay-The protein concentration was determined by the method of Bradford (25) using the Bio-Rad protein assay reagent according to the manufacturer's protocol with bovine serum albumin as a standard.
Preparation of Subcellular Fraction-All of the procedures were carried out at 4°C. Subcellular fractionation was performed by differential centrifugation according to previous studies (17,26) with some modifications. Three-day-old nondiapause eggs (1.0 g) were homogenized in 50 mM Tris-HCl buffer (2 ml), pH 7.5, containing 250 mM sucrose, 1 mM dithiothreitol, 0.5 mM PMSF, 10 M pepstatin A, and 10 M leupeptin. The homogenate was centrifuged at 1,000 ϫ g for 10 min to remove the chorion. The supernatant was centrifuged at 10,000 ϫ g for 30 min. The resulting supernatant was further centrifuged at 100,000 ϫ g for 60 min. A lipid layer obtained from centrifugation at 1,000 ϫ g was collected and homogenized to an emulsion. Each pellet (1,000 ϫ g pellet, 10,000 ϫ g pellet, and 100,000 ϫ g pellet), the lipid layer, and the final supernatant were used for the enzyme assay.
Purification of EPPase-All of the purification procedures were carried out at 4°C. Three-day-old nondiapause eggs (350 g) were used for enzyme purification. The eggs were homogenized in 2 volumes of 50 mM Tris-HCl buffer, pH 7.5, containing 250 mM sucrose, 1 mM dithiothreitol, 0.5 mM PMSF, 10 M pepstatin A, and 10 M leupeptin/g of eggs. An aliquot of 10,000 ϫ g supernatant was loaded onto a Q Sepharose HP column (Amersham Biosciences) pre-equilibrated with 50 mM Tris-HCl buffer, pH 7.5. Elution was performed using a linear gradient of 0 to 400 mM NaCl in 50 mM Tris-HCl buffer, pH 7.5, for 200 min at a flow rate of 6.5 ml/min. The active fraction was applied to a Chelating Sepharose FF column (Amersham Biosciences), which was previously charged with Zn 2ϩ by passing 0.1 M ZnCl 2 through the column and then equilibrated with 50 mM Tris-HCl buffer, pH 7.5, containing 500 mM NaCl.
Stepwise elution was performed using 50 mM EDTA in 50 mM Tris-HCl buffer, pH 7.5, containing 500 mM NaCl at a flow rate of 2.5 ml/min. The active fractions were collected, and NaCl was added to obtain a final concentration of 1.5 M for the following purification step. This enzyme solution was applied to a Phenyl Sepharose HP column (Amersham Biosciences) pre-equilibrated with 50 mM Tris-HCl buffer, pH 7.5, containing 1.5 M NaCl. Elution was performed using decreasing NaCl concentration at a flow rate of 2.0 ml/min. The active fraction was concentrated to about 10-fold, and the buffer was changed to 50 mM Tris-HCl buffer, pH 7.5, containing 1 mM MnCl 2 , 1 mM MgCl 2 , and 1 mM CaCl 2 using Centriplus 30 (Millipore). The enzyme solution was applied to a HiTrap ConA HP column (Amersham Biosciences) pre-equilibrated with the same buffer as the enzyme solution. The column was washed with 5 column volumes of the equilibration buffer, and then elution was performed using 0.5 M methyl-␣-D-glucoside in 50 mM Tris-HCl buffer, pH 7.5. The active fraction was applied to a Mono Q HR 5/5 column  (Amersham Biosciences) pre-equilibrated with 50 mM Tris-HCl buffer, pH 7.5. Elution was performed using a linear gradient of 0 -500 mM NaCl in 50 mM Tris-HCl buffer, pH 7.5, for 120 min at a flow rate of 1.0 ml/min. The active fraction was dialyzed against 50 mM Tris-HCl buffer, pH 7.5, and reapplied to the Mono Q column. The enzyme was eluted by a linear gradient of 0 -500 mM NaCl for 240 min. The active fraction was concentrated and loaded onto a Superdex 200HR 10/30 column (Amersham Biosciences). The enzyme was eluted with 50 mM Tris-HCl buffer, pH 7.5, containing 150 mM NaCl at a flow rate of 0.25 ml/min.
Kinetic Analysis-Besides E22P, other ecdysteroid-phosphates, 20E22P, 2-deoxyecdysone 22-phosphate (2dE22P) and 22-deoxy-20-hydroxyecdysone 3-phosphate (22d20E3P), which were isolated from mature ovaries of B. mori (27,28), were also used as substrates for the kinetic study of EPPase. Substrate concentration was varied from 0.4 to 204.8 M. Amounts of the products 20E and 2-deoxyecdysone formed in the reaction mixture were determined by RIA using antiserum N-6 as described in the previous section; this antiserum scarcely cross-reacted to their phosphoric esters (Ͻ0.058% in 20E22P and Ͻ0.019% in 2dE22P). 2 The amount of 22-deoxy-20-hydroxyecdysone (22d20E) converted from 22d20E3P was determined by RIA using antiserum H-22 (provided by Prof. L. I. Gilbert, University of North Carolina at Chapel Hill) without isolating 22d20E from 22d20E3P; cross-reactivity of 22d20E3P to this antiserum was below 0.5% of that of 22d20E. 2 Michaelis-Menten constants (K m and V max ) were graphically determined for each substrate from the Lineweaver-Burk plots. The turnover number 2 R. Yamada and H. Sonobe, unpublished data.  (k cat ) was calculated on the basis of molecular mass estimated by SDS-PAGE analysis.
Protein Sequencing-To obtain the internal amino acid sequence of EPPase, 1 nmol of reduced and S-calboxymethylated purified EPPase was digested with trypsin (sequencing grade; Sigma). The resulting tryptic peptides were separated by reverse-phase high performance liquid chromatography using a CAPCELL PAK C18 UG300 (2.0 ϫ 250 mm; Shiseido Fine Chemicals). Among the tryptic peptides, six peptides, named peptides 1-6, were sequenced by Edman degradation using an automated pulsed liquid phase protein sequencer (Applied Biosystems 476A).
cDNA Cloning and Sequencing-To clone the EPPase-cDNA fragment encoding peptides between peptides 1 and 5, a reverse transcription PCR was performed using degenerate primers. The forward primer was designed based on a part of the amino acid sequence of peptide 1 (EPP 1F, 5Ј-CA(A/G) GGI (C/T)TI CC(A/C/G/T) ATG AA(C/T) TGG, where I represents inosine). The reverse primer was designed based on a part of the amino acid sequence of peptide 5 (EPP 5R, 5Ј-(A/G)AA

(C/T)TC (A/G)TC CAT (A/C/G/T)GT (C/T)TC (A/C/G/T)GC).
For RNA extraction, the eggs were frozen in liquid N 2 at appropriate stages of embryonic development. Total RNA was extracted using Isogen (Nippongene) from 3-day-old nondiapause eggs according to the manufacturer's protocol. The first strand cDNA was reverse-transcribed from total RNA using a Ready-To-Go T-primed first strand cDNA synthesis kit (Amersham Biosciences) with the Not I d(T) primer (5Ј-AAC TGG AAG AAT TCG CGG CCG CAG GAA T 18 ). The synthesized first strand cDNA was subjected to PCR amplification with two degenerate primers: EPP 1F and EPP 5R. The PCR protocol was as follows: 1 cycle of 95°C for 5 min; 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min; and 1 cycle of 72°C for 10 min. The PCR products were separated by electrophoresis on a 1.5% agarose gel, and the objective product was purified using a Prep-A-Gene DNA Purification kit (Bio-Rad) according to the manufacturer's protocol. The purified PCR product was cloned into a pSTBlue-1 Acceptor vector (TaKaRa) by TA cloning. Transformation was screened by colony PCR using T7 and SP6 primers (5Ј-TAA TAC GAC TCA CTA TAG GG and 5Ј-CAT ACG ATT TAG GTG ACA CTA TAG, respectively). Double-stranded DNA sequencing was carried out by the dideoxy termination method using a BcaBEST dideoxy Sequencing kit (TaKaRa).
Rapid Amplification of 3Ј-and 5Ј-Ends-To obtain the 3Ј-end of the cDNA, the first strand cDNA synthesized as described above was used as a template for 30 cycles of the PCR amplification with a gene specific primer, GSP 1F (5Ј-CCC TGG GTG CCG CAC TGC TT) and an adopter primer (5Ј-AAC TGG AAG AAT TCG CGG), which is included in the Not I d(T) primer. The PCR protocol was as follows: 1 cycle of 95°C for 5 min; 30 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min; and 1 cycle of 72°C for 10 min. The PCR products were used as templates for the seminested PCR, which was carried out with a gene-specific primer GSP 2NF (5Ј-GTC GAC ATG ACT TAC AAA CCG) and an adopter primer under the following conditions: 1 cycle of 95°C for 5 min; 20 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min; and 1 cycle 72°C for 10 min.
To obtain the 5Ј-end of the cDNA, the first strand cDNA was synthesized from total RNA using a SMART RACE cDNA amplification kit (Clontech) according to the manufacturer's protocol. Two rounds of the PCR amplification performed using gene-specific primers GSP 3R and GSP 4NR (GSP 3R was 5Ј-CCC TCC TTT ACC ACC GGC TCT GTG CGC C, and GSP 4NR was 5Ј-CCC AAC GAC GCG ATT TTG AAG TAC CGT TCG) together with an anchor primer supplied by the manufacturer. The conditions for each cycle of the PCR were set according to the manufacturer's protocol.
Construction of cDNA Containing an Open Reading Frame-The cDNA containing the whole open reading frame of EPPase was amplified by the PCR from the first strand cDNA synthesized as described above. PCR was carried out using gene-specific primers (ORF F was 5Ј-ATA GGA TCC ATG GCC GGG ACA TAT CCT CAC, and ORF R was 5Ј-ACA AAG CTT TTA TTT TAT TAG TAT TCT CCA GTC G) under the following conditions: 1 cycle of 95°C for 5 min; 30 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 2 min; and 1 cycle 72°C for 10 min. The ORF F and ORF R primers were designed to incorporate BamHI and HindIII sites (underlined in the primer sequence), respectively. The PCR products were digested with the appropriate restriction enzymes and cloned into a pSTBlue-1 Acceptor vector. Five independent clones were sequenced.
Baculovirus Expression-Recombinant baculovirus harboring the EPPase-cDNA was constructed using the BAC-TO-BAC baculovirus expression system (Invitrogen) according to the manufacturer's protocol. The cDNA containing the whole open reading frame was cut with BamHI and HindIII from the plasmid as described above and ligated into the pFASTBAC plasmid. The recombinant plasmid was transformed in DH10BAC cells. The colonies containing recombinant bacmid DNA were selected by disruption of the lacZЈ gene and confirmed by colony PCR using a M13/pUC amplification primer (the forward primer was 5Ј-CCC AGT CAC GAC GTT GTA AAA CG, and the reverse primer was 5Ј-AGC GGA TAA CAA TTT CAC ACA GG). Spodoptera frugiperda cells (Sf9) were cultured in Sf900SFM (Invitrogen). The isolated recombinant bacmid DNA (1 g) was transfected to 1 ϫ   mM Tris-HCl buffer, pH 7.5, containing 250 mM sucrose, 1 mM dithiothreitol, 0.5 mM PMSF, 10 M pepstatin A, and 10 M leupeptin/g of cells. The homogenate was centrifuged at 10,000 ϫ g for 60 min at 4°C. The supernatant was loaded onto a HiTrap Q HP column (Amersham Biosciences) pre-equilibrated with 50 mM Tris-HCl buffer, pH 7.5. Elution was performed by a linear gradient of 0 -500 mM NaCl in 50 mM Tris-HCl buffer, pH 7.5, for 90 min at a flow rate of 1.0 ml/min. The active fraction was dialyzed against 50 mM Tris-HCl buffer, pH 7.5, and applied to a Mono Q HR column pre-equilibrated with the same buffer. Elution was performed using a linear gradient of 0 -500 mM NaCl in 50 mM Tris-HCl buffer, pH 7.5, for 120 min at a flow rate of 1.0 ml/min. The active fraction was concentrated and loaded onto a Superdex 200HR column. EPPase was eluted with 50 mM Tris-HCl buffer, pH 7.5, containing 150 mM NaCl at a flow rate of 0.25 ml/min. The purity was analyzed by SDS-PAGE (10% gel). The effluent was monitored at 280 nm.
Western Blot Analysis-Polyclonal antibodies against EPPase were raised in rabbits using the purified recombinant EPPase as an antigen. Antiserum from an immunized rabbit was purified using a HiTrap Protein A column (Amersham Biosciences) according to the manufacturer's protocol.
Northern Blot Analysis-Total RNA from various tissues was isolated using Isogen. Total RNA (20 g) was separated on a 1.0% formaldehyde-agarose gel, and the separated products were transferred to a Hybond-N ϩ nylon membrane (Amersham Biosciences) and hybridized with a probe corresponding to the whole open reading frame of the EPPase. The probe was labeled with alkaline phosphatase using the  ALKPhos Direct Labeling Module (Amersham Biosciences). Prehybridization and hybridization were carried out using ALKPhos Direct Hybridization Buffer (Amersham Biosciences) at 55°C. The blot was washed at 55°C with 150 mM sodium phosphate buffer, pH 7.5, containing 2 M urea, 0.1% SDS, 150 mM NaCl, and 1 mM MgCl 2 , and alkaline phosphatase-labeled bands were visualized using the CDP-Star Detection Reagent (Amersham Biosciences) as the substrate.

RESULTS
Biochemical Characterization of EPPase-To elucidate the subcellular localization of EPPase, the homogenate prepared from 3-day-old nondiapause eggs was fractionated by differential centrifugation. As shown in Fig. 2A, most of the EPPase activity was localized in the 100,000 ϫ g supernatant, suggesting that EPPase is a soluble enzyme. Acid phosphatase, a typical marker enzyme of lysosomes, was predominantly distributed in the 10,000 ϫ g pellet. The optimal pH levels of EPPase and acid phosphatase were 7.5 and 4.0, respectively (Fig. 2B). Fluoride and L-tartarate, which are strong inhibitors of lysosomal acid phosphatase (29 -31), scarcely affected the EPPase activity, although these chemicals also inhibited acid phosphatase in B. mori eggs (Fig. 2C). All of the results obtained indicate that EPPase apparently differs from lysosomal acid phosphatase.
Purification of EPPase-The 10,000 ϫ g supernatant prepared from 3-day-old nondiapause eggs was applied to a Q Sepharose HP column. Two peaks of the EPPase activity were eluted by a linear gradient of NaCl (Fig. 3A). The major peak, eluted within the concentration range from 170 to 200 mM (fractions 146 -158), was subjected to zinc-charged Chelating Sepharose column chromatography. Most of the EPPase activity was eluted when 50 mM EDTA was added to the equilibration buffer (Fig. 3B). The active fractions (fractions 44 -49) were pooled and applied to a Phenyl Sepharose column. Most of the EPPase activity was eluted with decreasing NaCl concentration (Fig. 3C). The active fractions (fractions 46 -52) were pooled and applied to a HiTrap ConA column. EPPase was not bound to the matrix (elution profile not shown), but about 67% of the contaminating proteins were removed at this step ( Table  I). The active fraction from the HiTrap ConA column was subjected to Mono Q column chromatography twice. In the first Mono Q column chromatography, the EPPase activity with a small shoulder peak was eluted by a linear gradient of NaCl. The major active fractions (fractions 34 -37) were pooled and resubjected to Mono Q column chromatography (Fig. 3E). The active fractions from the second Mono Q column chromatogra-phy (fractions 67-70) were pooled and subjected to gel filtration chromatography using a Superdex 200HR column, the EPPase activity was eluted as a single and symmetrical peak, and the peak corresponded to that monitored at 280 nm (Fig. 3F). SDS-PAGE analysis revealed that the protein obtained is a single protein with a molecular mass of 42 kDa (Fig. 3F, inset).
The results of purification at each step are summarized in Table I. After seven steps of column chromatography, 0.42 mg of EPPase was obtained from 350 g of 3-day-old nondiapause eggs with 1.3% yield, and the specific activity increased to 3,061-fold (Table I).
Kinetic Analysis of EPPase-To examine the substrate specificity of EPPase, first it was studied whether or not EPPase activity was inhibited by pNPP, which is generally used as a substrate of phosphatase (21)(22)(23)(24). As shown in Fig. 4, the EPPase activity was competitively inhibited by pNPP, indicat- ing that pNPP binds to the same catalytic site in EPPase as in the case of E22P. However, the specific constant (k cat /K m ) of EPPase was 16-fold lower for pNPP than for E22P (Table II). This result indicates that EPPase has greater specificity for E22P. Next, kinetic properties of the enzyme were analyzed for different ecdysteroid-phosphates in B. mori eggs. The k cat /K m value of the enzyme varied according to the substrate used (Table II). The k cat /K m values for 2dE22P and 20E22P were 1.4and 4.7-fold higher, respectively, than that for E22P. However, the k cat /K m value for 22d20E3P was about 200-fold lower than that for E22P. These results suggest that EPPase has greater specificity for ecdysteroid-phosphates that have a phosphate group at the C-22 position rather than at the C-3 position.
Two degenerate primers, EPP 1F and EPP 5R, were designed based on parts of amino acid sequences of peptides 1 and 5. These primers were used to amplify the EPPase-cDNA fragment (567 bp) by reverse transcription PCR using total RNA isolated from 3-day-old nondiapause eggs (Fig. 5). Gene-specific primers derived from this sequence were synthesized and used for 3Ј-and 5Ј-RACE. The 3Ј-RACE product (793 bp) contains a stop codon and an 18-bp poly(A) sequence, and the 5Ј-RACE product (414 bp) contains a putative translation start site (Fig.  5). Finally, a pair of gene-specific primers, ORF F and ORF R, were synthesized and used to amplify a cDNA fragment (996 bp) including the whole open reading frame of EPPase (Fig. 5).
The nucleotide sequence of this PCR product completely corresponded to that determined by 3Ј-and 5Ј-RACE.
The deduced amino acid sequence of the isolated cDNA contained each of the amino acid sequences of six peptides obtained by tryptic digestion of purified EPPase (Fig. 5). The full-length cDNA was composed of 1620 bp with an open reading frame encoding a protein of 331 amino acid residues (Fig. 5).
Expression of EPPase by Baculovirus Expression System-To confirm that the isolated cDNA encodes the functional EPPase, the recombinant EPPase was expressed using the baculovirus expression system, and its properties were compared with those of native EPPase. Sf9 cells were infected with recombinant baculovirus harboring the whole open reading frame of EPPase, and the cells were collected every 24 h after infection. The EPPase activity increased rapidly from 24 h after infection and reached a steady state 72 h after infection (Fig. 6). The EPPase activity was not detected in uninfected cells (Fig. 6). The recombinant EPPase was extracted from the cells infected at multiplicity of infection of 10 and purified according to the methods described under "Experimental Procedures." By Hi-Trap Q column chromatography, the recombinant EPPase was eluted within the NaCl concentration range from 170 to 210 mM (elution profile not shown). The active fraction was dialyzed and then subjected to Mono Q column chromatography. The recombinant EPPase was eluted within the NaCl concentration range from 150 to 170 mM (elution profile not shown). The active fraction was finally purified using a Superdex 200HR column (Fig. 7A). Western blot analysis of the recombinant and native EPPase using antibodies against recombinant EPPase showed that both proteins have the same antigenicity (Fig. 7B) and the same molecular mass (Figs. 3F and 7B). Furthermore,  (14). Diapause eggs cease to develop at the late gastrulation stage (14).
V max and K m values for E22P and pNPP of the recombinant EPPase (V max , 1.13 mol/min/mg protein and K m , 6.12 M for E22P; V max , 120.1 mol/min/mg protein and K m , 10613.0 M for pNPP) were almost the same as those of the native EPPase (Table II). These results indicate that the isolated cDNA encodes a functional EPPase.
Tissue Distribution and Expression Pattern of EPPase during Embryonic Development-Using a cDNA probe containing the whole open reading frame of EPPase, a transcript of ϳ1.7 kb was detected in 3-day-old nondiapause eggs but was not detected in diapause eggs (Fig. 8). Furthermore, the transcript was not detected in the mature ovary and testis at the pupal stage and Malpighian tubules, fat body, and midgut at the fifth instar of larvae.
The fluctuations of the amounts of free ecdysteroids and the EPPase activity during embryonic development are shown in Fig. 9A. In nondiapause eggs, the amounts of free ecdysteroids increased from 2 days after oviposition (late gastrula stage) and reached a peak 5 days after oviposition (blastokinesis stage). Thereafter, it decreased gradually as embryonic development proceeded. The EPPase activity increased slightly 1 day after oviposition (early gastrula stage) and reached a peak 3-4 days after oviposition (organogenesis stage). However, the activity decreased gradually to the basal level as embryonic development proceeded. In diapause eggs, the free ecdysteroids were detected only in trace amounts throughout the embryonic development. The EPPase activity was also not detected in diapause eggs. Northern blot analysis of the transcript during embryonic development is shown in Fig. 9B. In nondiapause eggs, the transcript was detected weakly 1 day after oviposition, and its signal intensified between days 2 and 5. However, after that it was scarcely detected. In diapause eggs, the transcript was not detectable throughout the embryonic development.

DISCUSSION
In eggs of various insect species, it has been suggested that maternal ecdysteroids are involved in cuticle formation during embryonic development (6 -9). However, evidence for this notion is based on temporal correlations rather than direct evidence. On the other hand, it was demonstrated that 20E is required for continuing to embryonic development in B. mori eggs; that is, the deficiency of 20E induces embryonic diapause (11,13). Furthermore, 20E was demonstrated to be produced both by de novo synthesis and dephosphorylation of maternal inactive ecdysteroid-phosphates (16 -18, 32). It was suggested that the hydroxylation at the C-20 position of E may be a late-limiting step of the de novo synthesis of 20E (17,32). Regarding the dephosphorylation of ecdysteroid-phosphates, there has been little information on the enzyme that specifically catalyzes dephosphorylation reaction not only in B. mori but also in other insect species, although lysosomal acid phosphatase prepared at the late stage of embryonic development has been reported to be capable of hydrolyzing E22P in S. gregaria (33,34).
EPPase found in the nondiapause eggs of B. mori had a cytosolic subcellular localization ( Fig. 2A) and was most active at pH 7.5 (Fig. 2B). The enzyme was unaffected by L-tartarate and fluoride, although acid phosphatase was inhibited by these inhibitors (Fig. 2C). The kinetic analysis of purified EPPase showed that although the affinity and specificity of EPPase for pNPP are much lower than those for E22P (Table II), both E22P and pNPP are hydrolyzed at the same active site of EPPase (Fig. 4). Therefore, there is no doubt that EPPase is a phosphatase. Furthermore, lysosomal acid phosphatase prepared from B. mori eggs scarcely hydrolyzed E22P, 2 and alkaline phosphatase in B. mori eggs could not be detected until 1.5 days before hatching (35). These results suggest that ecdysteroid-phosphates in B. mori eggs are exclusively hydrolyzed by EPPase. However, EPPase had only a very low specificity for 22d20E3P in comparison with E22P, 20E22P, and 2dE22P (Table II). This result suggests the possibility that EPPase has greater specificity for ecdysteroids whose side chain is phosphorylated. This notion is not contradictory with a hypothesis that ecdysteroids phosphorylated at C-22 position or C-26 position appear to be storage forms from which active ecdysteroids can be released, whereas ecdysteroids phosphorylated at C-2 position or C-3 position are true inactivation products of the hormone (34,36,37).
As shown in the data base Pfam (protein family of alignments) (www. sanger.ac.uk/Software/Pfam/imdex.shtml), phosphatases have been classified into many groups according to their consensus patterns of amino acid sequences, such as acid phosphatase, alkaline phosphatase, protein phosphatase, and fructose-1-6-bisphosphatase. In all sequences inputted in the Pfam data base, we could not find phosphatases that have identity, even in a part, with the amino acid sequence of EPPase. This result suggests that EPPase is a novel phosphatase that does not belong to already known phosphatase groups. In SilkBase, the expressed sequence tag data base of the silkworm B. mori (www.ab.a.u-tokyo.ac.jp/silkbase/), using tBLASTn analysis showed that the C-terminal region of EPPase (amino acids 170 -259) shares 93% identity with fbs2026 cloned from fat body of B. mori. The data base Swiss-Prot using FASTA analysis also showed that EPPase (amino acids 64 -329) has 38% identity with the C-terminal region of the UBASH3A protein homolog in Drosophila melanogaster (amino acids 489 -748). Although the functions of both proteins are not clarified at present, a possibility that these proteins are involved in ecdysteroid metabolism cannot be ruled out. Detailed analysis of the biochemical roles of these proteins will be necessary.
The pattern of changes in EPPase activity in diapause eggs and nondiapause eggs was nearly parallel to that of changes in the amounts of free ecdysteroids (Fig. 9A). This result is consistent with our previous results obtained from tracer experiments using [ 3 H]E22P and [ 3 H]20E22P (18), in which it has been demonstrated that the hydrolysis of ecdysteroid-phosphates is one of the effective causes leading to the increase in the amounts of free ecdysteroids. Furthermore, the pattern of changes in EPPase activity coincided with the changes in the expression pattern of the EPPase transcript (Fig. 9B), suggesting that gene transcript is required for eliciting the increase in EPPase activity. To investigate the general role of EPPase in embryonic development, we are currently attempting to clarify whether or not EPPase is involved in ecdysteroid metabolism in insects other than B. mori.