Ecdysteroid-phosphate phosphatase in silkworm eggs: purrification and characterization

在许多昆虫的卵中，蜕皮类固醇磷酸盐是母体起源的，已被广泛接受为胚胎发育过程中提供活性游离蜕皮类固醇的储存形式。然而，关于专门催化蜕皮类固醇-磷酸盐去磷酸化的酶的信息很少。本研究的目的是纯化和鉴定家蚕蜕皮激素-磷酸脱氢酶(EPPase)。已有研究表明，在家蚕胚胎发育的早期阶段，非滞育卵中蜕皮激素-磷酸脱磷反应比滞育卵中注射[~3H]蜕皮激素22-磷酸的活性高得多(Makka和Sonobe，2000)。因此，非滞育卵对EPPase的纯化是有用的。经蜕皮酮22-磷酸脱磷反应检测，EPPase活性定位于胞质组分。该酶通过七步柱层析纯化了约3,000倍的纯度。纯化后的酶在pH为7.5时酶活最高，不受溶酶体酸性磷酸酶强抑制剂酒石酸盐和氟化物的影响。所有其他酶的实验也表明，该酶不同于非特异性磷酸酶，对蜕皮类固醇磷酸具有高度的专一性。用RACE方法分离DNA末端。获得的全长CDNA全长为1620bp，编码331个氨基酸残基的蛋白质。将该蛋白与谷胱甘肽S转移酶融合，在SF9细胞中表达，并纯化至均一。融合蛋白具有EPPase活性。

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.