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Ecdysteroid-phosphate phosphatase in silkworm eggs: purrification and characterization

蚕卵中的脱皮素磷酸酶：纯化和表征

基本信息

批准号：
11640671
负责人：
SONOBE Haruyuki
金额：
$ 2.3万
依托单位：
Konan University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11640671/
关键词：
ecdysteroids Ecdysteroid-phosphate Phosphatase silkworm ホスファターゼ胚発生

项目摘要

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.

在许多昆虫的卵中，来源于母体的蜕皮甾体-磷酸盐已被广泛接受为在胚胎发育期间提供活性游离蜕皮甾体的储存形式。然而，很少有关于特异性催化蜕皮激素磷酸去磷酸化的酶的信息。本研究的目的是纯化和表征蜕皮甾体磷酸磷酸酶（EPPase）。在家蚕胚胎发育的早期阶段通过注射[3 H]蜕皮激素22-磷酸，已经证明非滞育卵中的蜕皮甾体磷酸的去磷酸化反应远比滞育卵中的去磷酸化反应活跃（Makka和Sonobe，2000）。因此，非滞育卵可用于纯化EPPP酶。通过蜕皮激素22-磷酸的去磷酸化反应测定的EPPase活性位于胞质溶胶组分中。该酶通过七步柱层析纯化约3，000倍至均一。纯化的酶在pH 7.5时最活跃，不受酒石酸盐和氟化物的影响，酒石酸盐和氟化物是溶酶体酸性磷酸酶的强抑制剂。利用RT-PCR技术，从3日龄非滞育卵中克隆到了EPPase的cDNA片段。RACE用于分离DNA的末端。获得的全长cDNA由1620 bp组成，具有开放阅读框，编码331个氨基酸残基的蛋白质。该蛋白与谷胱甘肽S-转移酶融合，在Sf 9细胞中表达并纯化至均一。融合蛋白具有EPPase活性。