| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1999: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
In eggs of many insect species, ecdysteroid-phosphates, which are of maternal origin, have widely been accepted as storage forms for supplying active free ecdysteroids during embryonic development. However, there is little information on the enzyme which specifically catalyzes the dephosphorylation of ecdysteroid-phosphates. The purpose of this study is to purify and characterize the ecdjysteroid-phosphate phosphatase (EPPase).
In the silkworm Bombyx morif it has been demonstrated that the dephosphorylation reaction of ecdysteroid-phosphates in non-diapause eggs is far more active than that in diapause eggs by injecting [3H]ecdysone 22-phosphate at early stages of embryogenesis (Makka and Sonobe, 2000). Therefore, non-diapause eggs are useful for purifying EPPase. EPPase activity, which was assayed by the dephosphorylation reaction of ecdysone 22-phosphate, was located in the cytosol fraction. This enzyme was purified about 3,000-fold to homogeneity by a seven-step column chromatography. The purified enzyme was most active at pH 7.5, and unaffected by tartrate and fluoride, which are strong inhibitors of lysosomal acid phosphatase. All other enzymic experiments also showed that the enzyme differs from non-specific phosphatase and possesses high specificity for ecdysteroid-phosphates.
Using RT-PCR, a CDNA fragment of EPPase was cloned from three-day-old non-diapause eggs. RACE was used to isolate the ends of the DNA. The full-length CDNA obtained was composed of 1620 bp with an open reading frame encoding a protein of 331 amino acid residues. The protein, fused with glutathione S-transferase, was expressed in Sf9 cells and purified to homogeneity. The fused protein had EPPase activity.
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