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• W2170695098 abstract "This is the first report succeeding in the isolation and characterization of an enzyme and its gene involved in the phosphorylation of a steroid hormone. It has been demonstrated that ecdysteroid 22-phosphates in insect ovaries, which are physiologically inactive, serve as a “reservoir” that supplies active free ecdysteroids during early embryonic development and that their dephosphorylation is catalyzed by a specific enzyme, ecdysteroid-phosphate phosphatase (Yamada, R., and Sonobe, H. (2003), J. Biol. Chem. 278, 26365–26373). In this study, ecdysteroid 22-kinase (EcKinase) was purified from the cytosol of the silkworm Bombyx mori ovaries to about 1,800-fold homogeneity in six steps of column chromatography and biochemically characterized. Results obtained indicated that the reciprocal conversion of free ecdysteroids and ecdysteroid 22-phosphates by two enzymes, EcKinase and ecdysteroid-phosphate phosphatase, plays an important role in ecdysteroid economy of the ovary-egg system of B. mori. On the basis of the partial amino acid sequence obtained from purified EcKinase, the nucleotide sequence of the cDNA encoding EcKinase was determined. The full-length cDNA of EcKinase was composed of 1,850 bp with an open reading frame encoding a protein of 386 amino acid residues. The cloned cDNA was confirmed to encode the functional EcKinase using the transformant harboring the open reading frame of EcKinase. A data base search showed that EcKinase has an amino acid sequence characteristic of phosphotransferases, in that it harbors Brenner's motif and putative ATP binding sites, but there are no functional proteins that share high identity with the amino acid sequence of EcKinase. This is the first report succeeding in the isolation and characterization of an enzyme and its gene involved in the phosphorylation of a steroid hormone. It has been demonstrated that ecdysteroid 22-phosphates in insect ovaries, which are physiologically inactive, serve as a “reservoir” that supplies active free ecdysteroids during early embryonic development and that their dephosphorylation is catalyzed by a specific enzyme, ecdysteroid-phosphate phosphatase (Yamada, R., and Sonobe, H. (2003), J. Biol. Chem. 278, 26365–26373). In this study, ecdysteroid 22-kinase (EcKinase) was purified from the cytosol of the silkworm Bombyx mori ovaries to about 1,800-fold homogeneity in six steps of column chromatography and biochemically characterized. Results obtained indicated that the reciprocal conversion of free ecdysteroids and ecdysteroid 22-phosphates by two enzymes, EcKinase and ecdysteroid-phosphate phosphatase, plays an important role in ecdysteroid economy of the ovary-egg system of B. mori. On the basis of the partial amino acid sequence obtained from purified EcKinase, the nucleotide sequence of the cDNA encoding EcKinase was determined. The full-length cDNA of EcKinase was composed of 1,850 bp with an open reading frame encoding a protein of 386 amino acid residues. The cloned cDNA was confirmed to encode the functional EcKinase using the transformant harboring the open reading frame of EcKinase. A data base search showed that EcKinase has an amino acid sequence characteristic of phosphotransferases, in that it harbors Brenner's motif and putative ATP binding sites, but there are no functional proteins that share high identity with the amino acid sequence of EcKinase. In steroid metabolism, it is generally accepted that cytochrome P450-mediated reaction, referred to as phase I metabolism, converts hydrophobic compounds into more polar metabolites, whereas phase II metabolism involves an adduct formation via a conjugation reaction. Phase II metabolism, such as sulfation, glucuronidation, and fatty acyl esterification, leads to a change of polarity or to a charge modification of the given metabolite, thereby modulating biological activity or facilitating elimination. In mammals, the sulfation pathway can be thought of as a reversible process, comprising two enzyme systems: the sulfotransferases, which catalyze the sulfation reaction, and the sulfatases, which catalyze the hydrolysis of sulfate esters formed by the action of sulfotransferases. Much is now known concerning the function of these enzyme systems as well as the molecular structure of the relevant cDNA and genes comprising these systems (1Weinshilboum R.M. Otterness D.M. Aksoy I.A. Wood T.C. Her C. Raftogianis R.B. FASEB J. 1997; 11: 3-14Crossref PubMed Scopus (392) Google Scholar, 2Hochberg R.B. Endocr. Rev. 1998; 19: 331-348Crossref PubMed Scopus (119) Google Scholar, 3Coughtrie M.W.H. Sharp S. Maxwell K. Innes N.P. Chem. Biol. Interact. 1998; 109: 3-27Crossref PubMed Scopus (189) Google Scholar, 4Raftogianis R. Creveling C. Weinshilboum R. Weisz J. J. Natl. Cancer Inst. Monogr. 2000; 27: 113-124Crossref PubMed Scopus (240) Google Scholar). However, to our knowledge, there exists very little information regarding the phosphorylation of steroid hormones in mammals. Ecdysteroids, which include the arthropod molting hormone, mediate a wide variety of developmental and reproductive events in insects (5Hagedorn H.H. Kerkut G.A. Gilbert L.I. Comprehensive Insect Physiology, Biochemistry and Pharmacology. 8. Pergamon, Oxford1985: 205-262Google Scholar). During the larval and pupal stages, ecdysteroids are synthesized in steroidogenic glands, known as the prothoracic glands (6Rybcznski R. Gilbert L.I. Iatrou K. Gill S.S. Comprehensive Molecular Insect Science. 3. Elsevier BV., Amsterdam2005: 61-123Google Scholar). The detailed ecdysteroid biosynthetic pathway constituted by phase I metabolism is not fully clarified, but cytochrome P450 enzymes catalyzing the last four steps of the biosynthesis of 20-hydroxyecdysone (the principal molting hormone of arthropods) (i.e. CYP306a1 (25-hydroxylase), CYP302a1 (22-hydroxylase), CYP315a1 (2-hydroxylase), and CYP314a1 (20-hydroxylase)), have been identified and characterized in Drosophila melanogaster, Bombyx mori, and Aedes aeypti (7Chavez V.M. Marques G. Delbeque J.P. Kobayashi K. Hollingsworth M. Burr J. Natzle J.E. O'Connor M.B. Development. 2000; 127: 4115-4126PubMed Google Scholar, 8Warren J.T. Petryk A. Marques G. Jarcho M. Parvy J.-P. Dauphin-Villemant C. O'Connor M.B. Gilbert L.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11043-11048Crossref PubMed Scopus (262) Google Scholar, 9Petryk A. Warren J.T. Marques G. Jarcho M.P. Gilbert L.I. Kahler J. Parvy J.P. Li Y. Dauphin-Villemant C. O'Connor M.B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13773-13778Crossref PubMed Scopus (335) Google Scholar, 10Niwa R. Matsuda T. Yoshiyama T. Namiki T. Mita K. Fijimoto Y. Kataoka H. J. Biol. Chem. 2004; 279: 35942-35949Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 11Warren J.T. Petryk A. Marques G. Parvy J.-P. Shinoda T. Itoyama K. Kobayashi J. Jarcho M. Li Y. O'Connor M.B. Dauphin-Villemant C. Gilbert L.I. Insect Biochem. Mol. Biol. 2004; 34: 991-1010Crossref PubMed Scopus (220) Google Scholar, 12Sieglaff D.H. Duncan K.A. Brown M.R. Insect Biochem. Mol. Biol. 2005; 35: 471-490Crossref PubMed Scopus (61) Google Scholar, 13Gilbert L.I. Mol. Cell. Endocrinol. 2004; 215: 1-10Crossref PubMed Scopus (219) Google Scholar). Although glucosides (14Warren J.T. Steiner B. Dorn A. Pak M. Gilbert L.I. J. Liq. Chromatogr. 1986; 9: 1759-1782Crossref Scopus (53) Google Scholar), fatty acyl esters (15Lanot R. Connat J.-L. Koolman J. Ecdysone. Georg Thieme Verlag, Stuttgart, Germany1989: 262-270Google Scholar), and sulfate esters (16Matsumoto E. Matsui M. Tamura H. Biosci. Biotechnol. Biochem. 2003; 67: 780-1785Crossref Scopus (5) Google Scholar) of ecdysteroids are detected as minor products of phase II reaction in several insect species, the major products of the phase II reaction in most insect species are phosphate esters (17Lafont R. Dauphin-Villemant C. Warren J.T. Rees H. Gilbert L.I. Iatrou K. Gill S.S. Comprehensive Molecular Insect Science. 3. Elsevier BV., Amsterdam2005: 125-195Google Scholar, 18Rees H.H. Isaac R.E. Hoffmann J.A. Porchet M. Biosynthesis and Mode of Action of Invertebrate Hormones. Springer, Berlin1984: 181-195Google Scholar, 19Koolman J. Zool. Sci. 1990; 7: 563-580Google Scholar, 20Lafont R. Harmatha J. Marion-Poll F. Dinan L. Wilson I.D. The Ecdysone Handbook. 3rd Ed. 2002http://ecdybase.orgGoogle Scholar). The ovaries of most insect species have the capacity to accumulate ecdysteroid phosphates, which are physiologically inactive (21Makka T. Seino A. Tomita S. Fujiwara H. Sonobe H. Arch. Insect Biochem. Physiol. 2002; 51: 111-120Crossref PubMed Scopus (47) Google Scholar), in addition to the capacity to synthesize free ecdysteroids de novo (22Kappler C. Goltzene F. Lagueux M. Hetru C. Hoffmann J.A. Int. J. Invert. Reprod. Dev. 1986; 9: 17-34Crossref Scopus (33) Google Scholar, 23Sonobe H. Tokushige H. Makka T. Hara N. Fujimoto Y. Zool. Sci. 1999; 16: 935-943Crossref Scopus (20) Google Scholar). It has been suggested that the ecdystreoid phosphates that are accumulated in the ovaries are transferred to the eggs and function as a source of active free ecdysteroid before the prothoracic glands differentiate during embryonic development (24Lafont R. Koolman J. Hoffmann J.A. Porchet M. Biosynthesis and Mode of Action of Invertebrate Hormones. Springer, Berlin1984: 196-226Google Scholar, 25Hoffmann J.A. Lagueux M. Kerkut G.A. Gilbert L.I. Comprehensive Insect Physiology, Biochemistry, and Pharmacology. 1. Pergamon, Oxford1985: 435-460Google Scholar, 26Rees H.H. Eur. J. Entomol. 1995; 92: 9-39Google Scholar). Recently, in B. mori eggs, a novel enzyme ecdysteroid-phosphate phosphatase (EPPase) 3The abbreviations used are: EPPase, ecdysteroid-phosphate phosphatase; EcKinase, ecdysteroid 22-kinase; HPLC, high performance liquid chromatography; RT, reverse transcription; RACE, rapid amplification of cDNA ends; EST, expressed sequence tag. 3The abbreviations used are: EPPase, ecdysteroid-phosphate phosphatase; EcKinase, ecdysteroid 22-kinase; HPLC, high performance liquid chromatography; RT, reverse transcription; RACE, rapid amplification of cDNA ends; EST, expressed sequence tag. (Fig. 1), which is specifically involved in dephosphorylation of ecdysteroid phosphates, was isolated, characterized, and revealed to be a vital enzyme that may control the “on/off switch” of embryonic development (27Yamada R. Sonobe H. J. Biol. Chem. 2003; 278: 26365-26373Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 28Yamada R. Yamahama Y. Sonobe H. Zool. Sci. 2005; 22: 187-198Crossref PubMed Scopus (31) Google Scholar, 29Sonobe H. Yamada R. Zool. Sci. 2004; 21: 503-516Crossref PubMed Scopus (72) Google Scholar). The discovery of EPPase in B. mori eggs emphasizes the physiological significance of phase II phosphorylation reaction in the ovary-egg system of insects. However, detailed characterization of enzymes involved in the formation of ecdysteroid phosphates has not been carried out, although the occurrence of enzyme activity has been reported in the ovaries of Schistocereca gregaria (30Kabbouh M. Rees H.H. Insect Biochem. 1991; 21: 57-64Crossref Scopus (9) Google Scholar) and B. mori (31Takahashi S.Y. Okamoto K. Sonobe H. Kamba M. Ohnishi E. Zool. Sci. 1992; 9: 169-174Google Scholar). We now report the purification, kinetic characterization, and cDNA cloning of an enzyme responsible for phosphorylation at the C-22 position of ecdysteroids in the mature oocytes of B. mori, named as ecdysteroid 22-kinase (EcKinase) (Fig. 1), and show that EcKinase belongs to a novel kinase family. Experimental Animals—The pupae of a hybrid race (kinshu x showa) of the silkworm B. mori, which have been programmed to lay diapause eggs (diapause egg-producing pupae), were mainly employed. In order to obtain pupae programmed to lay nondiapause eggs (nondiapause egg-producing pupae), the subesophageal ganglion, the source of diapause hormone, was removed soon after larval-pupal ecdysis (32Fukuda S. Proc. Jpn. Acad. 1951; 27: 672-677Crossref Google Scholar, 33Sonobe H. Ohnishi E. Dev. Growth Differ. 1970; 12: 41-52Crossref PubMed Scopus (25) Google Scholar). Pupal-adult ecdysis in both types of pupae took place 12–13 days after the larval-pupal ecdysis at 23–24 °C. There was no appreciable difference in the growth rate of ovaries between both types of pupae. Chemicals—[23,24-3H]Ecdysone (2,112 GBq/mmol) was obtained from PerkinElmer Life Sciences. Ecdysone and 20-hydroxyecdysone were purchased from Sigma. Ecdysone 22-phosphate was synthesized chemically (34Yamada R. Sonobe M. Tatara A. Fujimoto Y. Sonobe H. Keller R. Dircksen H. Sedlmeier D. Vaudry H. Conference of European Comparative Endcrinologists. Monduzzi Editore, Bologna, Italy2002: 179-183Google Scholar). Other ecdysteroids, 2-deoxyecdysone, 20-hydroxyecdysone, 22-deoxy-20-hydroxyecdysone, and 2,22-dideoxy-20-hydroxyecdysone, were extracted from B. mori ovaries and purified by high performance liquid chromatography (HPLC), as described previously (35Ohnishi E. Mizuno T. Ikekawa N. Ikeda T. Insect Biochem. 1981; 11: 55-159Crossref Scopus (43) Google Scholar, 36Ohnishi E. Hiramoto M. Fujimoto Y. Kakinuma K. Ikekawa N. Insect Boichem. 1989; 19: 95-101Crossref Scopus (28) Google Scholar, 37Kamba M. Mamiya Y. Sonobe H. Fujimoto Y. Insect Biochem. Mol. Biol. 1994; 24: 395-402Crossref Scopus (21) Google Scholar). Preparation of Enzyme Solution—All operations were carried out at ∼4 °C. For subcellular fractionation, mature ovaries were excised from adult moths in insect Ringer's solution, and their chorions were removed using tweezers in a small volume of homogenization buffer: 10 mm Hepes-NaOH buffer, pH 7.5, containing 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 10 μm pepstatin A, and 10 μm leupeptin. The ooplasm obtained did not contain follicle cells and nurse cells as observed by light microscopy. The ooplasm was centrifuged at 1,000 × g for 5 min. The resultant pellet (yolk granule fraction) was washed again with the same buffer solution and centrifuged, and the pellet was used as the yolk granule fraction. The supernatant was combined, homogenized, and used for differential centrifugation, the procedure of which is essentially the same as that described in our previous paper (38Horike N. Sonobe H. Arch. Insect Biochem. Physiol. 1999; 41: 9-17Crossref PubMed Scopus (29) Google Scholar). The homogenate was first centrifuged at 1,000 × g for 30 min, and the resultant supernatant was further centrifuged at 10,000 × g for 30 min. The next resultant supernatant was further centrifuged at 150,000 × g for 60 min. The yolk granule fraction, 1,000 × g pellet, 10,000 × g pellet, 150,000 × g pellet, and the lipid layer obtained from centrifugation at 10,000 × g were sonicated in a small volume of homogenization buffer to an emulsion state. These emulsions and the final 150,000 × g supernatant (cytosol) were used for the enzyme assay. To determine changes in enzyme activity during ovarian development, ovaries during pupal-adult development were dissected and homogenized with ∼5 volumes of the homogenization buffer. The homogenate was centrifuged immediately at 150,000 × g for 60 min. The supernatant was used as the enzyme source. For enzyme purification, mature ovaries (450 g) from diapause egg-producing pupae were employed. The ovaries were ground using an agate mortar in ∼2 volumes of homogenization buffer, and the homogenate was filtered through gauze to remove chorions. The filtrate was further homogenized with a glass-Teflon homogenizer, and the homogenate was centrifuged at 150,000 × g for 60 min. The resultant supernatant was applied to column chromatography (see below). Enzyme Assay—The activity of ecdysone 22-kinase, which was subsequently named EcKinase (see “Results”), was measured using [3H]ecdysone as the substrate. The standard assay system for ecdysone 22-kinase activity contains the following in 100 μl of 10 mm Hepes-NaOH buffer, pH 7.5, containing 0.1% bovine serum albumin: 2 mm ATP, 10 mm MgCl2, 740 Bq of [3H]ecdysone, and enzyme solution. The reaction was initiated by adding the enzyme solution, the reaction mixture was incubated at 35 °C for 30 min, and the reaction was stopped by adding 4 volumes of methanol. The mixture was centrifuged at 10,000 × g for 10 min at 4 °C. The supernatant was collected and evaporated to dryness under vacuum. The residue was dissolved in 1 ml of distilled water and applied to a C18 cartridge (Varian). After washing with 4 ml of distilled water to remove ATP and any salts, ecdysteroids were eluted with 6 ml of methanol. The ecdysteroid fraction was dried, and the residue was dissolved in 1 ml of 70% (v/v) chloroform/methanol and applied to a silicic acid cartridge (Varian). After washing with 5 ml of 70% chloroform/methanol to remove unreacted [3H]ecdysone, [3H]ecdysone 22-phosphate was eluted with 6 ml of 80% (v/v) methanol/water. The radioactivity of the [3H]ecdysone 22-phosphate fraction was measured using a liquid scintillation counter (LSC-5101; Aloka) to quantify ecdysone 22-phosphate formed. The recovery of radioactivity throughout the entire procedure was more than 90%. For the characterization of substrate specificity of ecdysone 22-kinase, 20-hydroxyecdysone, 2-deoxyecdysone, 2-deoxy-20-hydroxyecdysone, 22-deoxy-20-hydroxyecdysone, and 2,22-dideoxy-20-hydroxyecdysone were used in addition to ecdysone. Enzyme activity was assayed using the standard assay system for ecdysone 22-kinase activity by substituting the above mentioned ecdysteroids (20 μm) for the usual substrate, [3H]ecdysone. Following HPLC (see below), the reaction products were monitored at 254 nm and quantified using an on-line integrator D-2500 (Hitachi). The relative activity of the enzyme was expressed by defining the amount of ecdysone 22-phosphate produced in the control assay as 100%. For the identification of the products of enzyme reactions, the ecdysteroid phosphate fraction was divided into two portions. One portion was used to compare retention times with those of authentic ecdysteroid phosphates by HPLC (see below). The remainder was used in EPPase hydrolysis; the retention times of free ecdysteroids liberated after EPPase hydrolysis were compared with those of authentic free ecdysteroids. A competition assay was performed by adding ecdysteroids at a concentration of 100 μm as a competitor into the standard assay system. The radioactivity of [3H]ecdysone 22-phosphate produced was compared with the control without the competitor. The inhibitory effect was expressed as a percentage of control. The activity of the recombinant EcKinase was measured using the standard assay system by substituting 20 μm ecdysone for the usual substrate, [3H]ecdysone. The ecdysteroid fraction in the reaction mixture was applied to HPLC (see below), and the effluent was monitored at 254 nm. Protein Assay—Protein concentration was determined by the method of Bradford (39Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215535) Google Scholar), using the Bio-Rad protein assay reagent (Bio-Rad) according to the manufacturer's protocol with bovine serum albumin as a standard. HPLC Analysis of Ecdysteroids—Ecdysteroid phosphates and free ecdysteroids liberated by enzymatic hydrolysis (see below) were identified using HPLC (LC-10A system; Shimazu) equipped with a reverse-phase column (4.6 × 250 mm; Wakosil 5C18; Wako, Japan). The column was eluted with a 50- or 60-min linear gradient of methanol in 20 mm potassium phosphate buffer, pH 5.6, changing from 0 to 70% (v/v) at a flow rate of 1 ml/min at 40°. Quantification of Ecdysteroid Phosphates in Ovaries—Ecdysteroids were extracted from ovarian homogenate by adding 4 volumes of methanol, and ecdysteroid phosphates were separated from free ecdysteroids as described previously (37Kamba M. Mamiya Y. Sonobe H. Fujimoto Y. Insect Biochem. Mol. Biol. 1994; 24: 395-402Crossref Scopus (21) Google Scholar). To enhance the sensitivity to radioimmunoassay using the antiserum N6 (40Nakatsuji T. Sonobe H. Gen. Comp. Endocrinol. 2004; 135: 358-364Crossref PubMed Scopus (88) Google Scholar), ecdysteroid phosphates were hydrolyzed to free ecdysteroids, using a digestive enzyme preparation from Helix pomatia (see below). The radioimmunoassay was conducted as described previously (41Sonobe H. Kamba M. Ohta K. Ikeda M. Naya Y. Experientia. 1991; 47: 948-952Crossref Scopus (48) Google Scholar). Ecdysteroid quantity was expressed in terms of ecdysone equivalents. Enzymatic Hydrolysis of Ecdysteroid Phosphates—Ecdysteroid phosphates were hydrolyzed to free ecdysteroids by recombinant EPPase (27Yamada R. Sonobe H. J. Biol. Chem. 2003; 278: 26365-26373Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) or the H. pomatia digestive enzyme preparation (Nakarai Tesque). The former specifically catalyzes the dephosphorylation of ecdysteroid 22-phosphate, whereas the latter contains nonspecific phosphatase activity. The procedures of each enzymatic hydrolysis have been described previously (27Yamada R. Sonobe H. J. Biol. Chem. 2003; 278: 26365-26373Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 28Yamada R. Yamahama Y. Sonobe H. Zool. Sci. 2005; 22: 187-198Crossref PubMed Scopus (31) Google Scholar, 37Kamba M. Mamiya Y. Sonobe H. Fujimoto Y. Insect Biochem. Mol. Biol. 1994; 24: 395-402Crossref Scopus (21) Google Scholar). Purification Procedures of Ecdysone 22-Kinase—All of the purification procedures were carried out at ∼4 °C. An aliquot of the 150,000 × g supernatant prepared from mature ovaries (see “Preparation of Enzyme Solution”) was loaded onto a blue Sepharose 6 FF column (26 × 75 mm; Amersham Biosciences) pre-equilibrated with 10 mm Hepes-NaOH buffer, pH 7.5. Stepwise elution was performed using the same buffer, containing 2 m KCl at a flow rate of 4 ml/min. The active fraction was applied to a chelating Sepharose FF column (26 × 75 mm; Amersham Biosciences) pre-equilibrated with 10 mm Hepes-NaOH buffer, pH 7.5, containing 2 m KCl. Stepwise elution was performed using the same buffer, containing 100 mm histidine at a flow rate of 4 ml/min. The active fraction was loaded onto a Sephacryl S-100 HR column (26 × 600 mm; Amersham Biosciences) pre-equilibrated with 10 mm Hepes-NaOH buffer, pH 7.5, containing 150 mm NaCl. The enzyme was eluted with the same buffer at a flow rate of 2 ml/min. The active fraction was concentrated to about 50-fold, and the buffer was changed to 5 mm Tris-HCl buffer, pH 7.5, using an Amicon Ultra-15 centrifugal filter device (Millipore Corp.). The enzyme solution was applied to a Mono Q HR 5/5 column (Amersham Biosciences) pre-equilibrated with 5 mm Tris-HCl buffer, pH 7.5. Elution was performed using a linear gradient of 0–100 mm NaCl in the same buffer for 20 min at a flow rate of 1 ml/min. The active fraction was concentrated to about 100-fold, and the buffer was changed to 10 mm Hepes-NaOH buffer, pH 7.5. The enzyme solution was applied to a ceramic hydroxyapatite column (Type 1, 7 × 52 mm; Bio-Rad) pre-equilibrated with 10 mm Hepes-NaOH buffer, pH 7.5. Elution was performed by changing the equilibration buffer linearly to 200 mm potassium-phosphate buffer, pH 7.5, for 90 min at a flow rate of 1 ml/min. The active fraction was dialyzed against 5 mm Tris-HCl buffer, pH 7.5, and applied again to the Mono Q HR 5/5 column pre-equilibrated with 5 mm Tris-HCl buffer, pH 7.5. The enzyme was eluted by a linear gradient of 0–100 mm NaCl for 45 min. The purity was analyzed using HPLC equipped with a reverse-phase column (CAPCELL PACK C18, 2 × 250 mm; Shiseido Fine Chemicals) pre-equilibrated with 0.05% trifluoroacetic acid. Elution was performed using a 30-min linear gradient of 0–80% acetonitrile in 0.05% trifluoroacetic acid at a flow rate of 1 ml/min at 40 °C. The effluent was monitored at 280 nm. The purity was also analyzed by SDS-PAGE, using a 10% gel. Several standard proteins (e.g. myosin (205 kDa), phosphorylase b (97.4 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), and trypsin inhibitor (21.5 kDa)) were used to estimate the molecular mass of purified ecdysone 22-kinase. The proteins were visualized by silver staining. Amino Acid Sequence Analysis—In order to analyze the internal amino acid sequence of purified ecdysone 22-kinase, 500 pmol of purified enzyme were reduced with dithiothreitol and alkylated with iodoacetamide and digested with lysylen-dopeptidase. The resultant peptides were applied to reverse-phase HPLC using a TSK gel ODS-80Ts (2.0 × 250 mm; TOSOH) pre-equilibrated with 0.1% trifluoroacetic acid. Elution was performed with a 90-min linear gradient of 0–90% acetonitrile in 0.09% trifluoroacetic acid at a flow rate of 0.4 ml/min. Among the peptides obtained, four peptides, named K54, K71, K75-1, and K75-2, were applied to an amino acid sequencer (see below), and one peptide, named K94, was digested again with trypsin. The digests were separated by reverse-phase HPLC using a Symmetry C18 column (1.0 × 150 mm; Waters) pre-equilibrated with 2% acetonitrile containing 0.1% trifluoroacetic acid. Elution was performed with a 100-min linear gradient of 2–90% acetonitrile in 0.1% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 0.05 ml/min. Two fragments obtained, named K94-T1 and K94-T2, were applied to an amino acid sequencer. All amino acid sequence analyses were performed using a Procise 494 cLC protein sequencing system (Applied Biosystems). Isolation of EcKinase cDNA—The 12–40-h-old diapause eggs of strain p50T were collected, and total RNA was prepared using TRIzol Reagent (Invitrogen). Construction of the full-length cDNA library, named fdpe, was carried out by Hitachi Instruments Service Co., Ltd. (Tokyo, Japan). Sequences from the 5′-end of each cDNA were determined, and the expressed sequence tags (ESTs) were compiled into a local data base. One EST clone, which is expected to encode the amino acid sequences of six peptides obtained from purified EcKinase, was searched by means of standard BLAST analysis, and the full length of its nucleotide sequence was determined. 5′-Rapid Amplification of cDNA Ends (5′-RACE)—Total RNA was prepared from mature ovaries by the same method as described above. First-strand cDNA was synthesized using a SMART RACE cDNA amplification Kit (Clontech) according to the protocol provided by the manufacturer. Two gene-specific primers, R1 (5′-TCGGTGTCGTACACGCTGTTGATG-3′) and R2 (5′-CATGAGGTTGCAGGTCCTGTAGTC-3′), were designed based on the nucleotide sequence of one EST clone, named fdpeP12_F_I09. The 5′-end of EcKinase cDNA was amplified by two rounds of PCR. In the first PCR, amplification was performed with R2 and a 5′-adapter primer, UPM (Clontech), using the first-strand cDNA as a template. PCR was carried out as follows: first denaturation at 95 °C for 30 s; five cycles of 95 °C for 10 s, 68 °C for 10 s, 72 °C for 2 min; five cycles of 95 °C for 10 s, 66 °C for 10 s, 72 °C for 2 min; 35 cycles of 95 °C for 10 s, 64 °C for 10 s, 72 °C for 2 min; and final extension at 72 °C for 2 min. In the second round of PCR, amplification was performed with R1 and a 5′-nested adapter primer, NUP (Clontech), using PCR product obtained in the first PCR as a template. The following program was used for PCR amplification: first denaturation at 95 °C for 30 s; five cycles of 95 °C for 10 s, 68 °C for 10 s, 72 °C for 2 min; five cycles of 95 °C for 10 s, 66 °C for 10 s, 72 °C for 2 min; 20 cycles of 95 °C for 10 s, 64 °C for 10 s, 72 °C for 2 min; and final extension at 72 °C for 2 min. Detection of EcKinase Transcript—Total RNA was prepared from mature ovaries and 72-h-old diapause eggs according to the same methods as described above. Each first-strand cDNA was synthesized with random hexamer using Ready-to-Go RT-PCR Beads (Amersham Biosciences) according to the manufacturer's instructions. The following primers were designed to detect the expression of EcKinase and elongation factor 2 as an internal control: F1 (5′-AAGCACTGGTGCTGGAGGACCTG-3′) and R3 (5′-TGCTGGCTCCTGTCATCCACTATC-3′) for EcKinase, EF2-F (5′-GTCGTACCGTGAGACCGTAGCTG-3′) and EF2-R (5′-CAATGTCCTCTGGCAGACCATCAG-3′) for elongation factor 2. The first strand cDNA was used as templates, and each PCR amplification was conducted by the following program: first denaturation at 94 °C for 30 s; 45 cycles of 94 °C for 20 s, 64 °C for 20 s, 72 °C for 1 min 30 s; and final extension at 72 °C for 1 min 30 s. PCR products were electrophoresed by an 1.5% agarose gel and visualized after staining with ethidium bromide. Construction of the EcKinase Expression Plasmid—In order to avoid the artificial effects of additional sequences, such as His tag and/or some epitope tags, which are encoded in commercial expression plasmids, on biological activity of the recombinant enzyme, we constructed the expression plasmid to express the whole open reading frame of EcKinase without artificial sequences according to the following strategy. The forward primer (EF, 5′-AAGCTTGACATGACGGACACCGAGGAGCG-3′) contained the HindIII site (italicized in the primer sequence) and 23 residues (nucleotide positions 29–51 in Fig. 10), including the initiation Met codon (boldfaced in the primer sequence). The reverse primer (ER, 5′-GAATTCTCAGATGAGACCCCAGCGGACGT-3′) contained the EcoRI site (italicized in the primer sequence) and 23 residues (nucleotide positions 1,170–1,192 in Fig. 10), including the stop codon (boldfaced in the primer sequence). Using these primers, PCR was conducted with the EST clone, fdpeP12_F_I09, as the template. The amplified cDNA insert was subcloned into pCR2.1-TOPO plasmids (Invitrogen), and the sequence fidelity was confirmed. Subsequently, the insert was released from pCR2.1-TOPO by HindIII/EcoRI digestion and then ligated into the HindIII/EcoRI site of the expression plasmid pIB/V5-His (Invitrogen). Expression of the EcKinase Cons" @default.
• W2170695098 created "2016-06-24" @default.
• W2170695098 creator A5008086549 @default.
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• W2170695098 date "2006-10-01" @default.
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• W2170695098 title "Purification, Kinetic Characterization, and Molecular Cloning of a Novel Enzyme, Ecdysteroid 22-Kinase" @default.
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• W2170695098 doi "https://doi.org/10.1074/jbc.m604035200" @default.
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