Ecdysteroid-phosphate phosphatase in silkworm eggs: purrification and characterization

在许多昆虫物种的卵中，卵磷脂被广泛认为是胚胎发育过程中提供活性游离卵磷脂的储存形式。然而，很少有关于这种酶的信息，这种酶能特异性地催化蜕皮甾体-磷酸盐的去磷酸化。本研究的目的是纯化和表征胆囊类固醇-磷酸磷酸酶（EPPase）。在家蚕（Bombyx morif）中，通过在胚胎发生早期注射[3H]蜕皮激素22-磷酸，已经证明非滞育卵中蜕皮激素-磷酸盐的去磷酸化反应远比滞育卵活跃（Makka和Sonobe， 2000）。因此，非滞育卵对纯化EPPase是有用的。通过22-磷酸蜕皮酮的去磷酸化反应测定了EPPase的活性，发现其位于细胞质部分。该酶通过七步柱层析纯化约3000倍至均匀性。纯化后的酶在pH为7.5时最具活性，不受酒石酸盐和氟化物的影响，这两种物质是溶酶体酸性磷酸酶的强抑制剂。所有其他酶实验也表明，该酶不同于非特异性磷酸酶，对体外甾体磷酸酯具有高特异性。利用RT-PCR技术，从3日龄非滞育卵中克隆了EPPase CDNA片段。RACE用于分离DNA的末端。CDNA全长1620 bp，开放阅读框编码331个氨基酸残基的蛋白。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匀性。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.