Biochemical characterization of small GTP binding protein in the brain of insect

昆虫大脑中小 GTP 结合蛋白的生化特征

• 批准号: 18580055
• 负责人: UNO Tomohide
• 金额: $ 1.92万
• 依托单位: Kobe University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18580055/
• 关键词: insect; brain; small GTP binding protein; antibody; Bombyx mori; rab; リン酸化

Small GTPases of the Rab proteins are key regulators of membrane trafficking. It was reported that rab 8 from Bombyx mori (BRab8) was phosphorylated by protein kinase C (PKC), which were expressed in mammalian cells and partially purified (Uno, et. al., 2006). To determine the phosphorylated amino acid residues of BRab8, seven serine and four threonin residues were shown as candidates to be phosphorylated. So, six deleted or mutated fragments containing cDNAs for BRab8 were constructed using PCR. The obtained fragments were inserted into expression vectors. Fusion-proteins were expressed in E.coli and partially purified using affinity chromatographies. These proteins were phosphorylated by PKC and gamma ^<32>P-ATP in vitro, electrophoresed and analyzed by a bioimaging analyzer. As a result, one serine-132 of BRab8 was specifically phosphorylated. Further, polyclonal antibody was isolated by immunizing with rab8 protein. From immunoblotting and staining phosphorylated proteins, Rab8 was found to be phosphorylated in vivo.

Rab 蛋白的小 GTP 酶是膜运输的关键调节因子。据报道，来自家蚕的 rab 8 (BRab8) 被蛋白激酶 C (PKC) 磷酸化，蛋白激酶 C (PKC) 在哺乳动物细胞中表达并部分纯化 (Uno, et. al., 2006)。为了确定 BRab8 的磷酸化氨基酸残基，七个丝氨酸和四个苏氨酸残基被显示为待磷酸化的候选残基。因此，使用 PCR 构建了 6 个含有 BRab8 cDNA 的缺失或突变片段。将获得的片段插入表达载体中。融合蛋白在大肠杆菌中表达并使用亲和层析进行部分纯化。这些蛋白质在体外被PKC和γ 32 P-ATP磷酸化、电泳并通过生物成像分析仪进行分析。结果，BRab8的1个丝氨酸132被特异性磷酸化。此外，通过用rab8蛋白免疫来分离多克隆抗体。通过免疫印迹和磷酸化蛋白染色，发现 Rab8 在体内被磷酸化。

项目成果

Rab GTPase, small GTP binding proteins, from the brain of Bombyx mon

Rab GTPase，小 GTP 结合蛋白，来自家蚕大脑

• 发表时间: 2008
• 作者: Keisuke;Hata;Le;Thi Dieu Trang;Tsubasa;Moriwaki;Takuya;Nakada;Susumu;Hiragaki;Michihiro;Takagi;Kengo;Kanamaru;Hiroshi;Yamagata;Makio;Takeda;Tomohide Uno
• 通讯作者: Tomohide Uno

Bioconversion by functional P450 .1A9 and IClof Anguillus japonica

通过功能性 P450 .1A9 和 IClof Anguillus japonica 进行生物转化

• 发表时间: 2007
• 期刊: Comprehensive Biochemistry and Physiology C 147
• 作者: Uno;T;Okamoto;S;Masuda;S;Imaisihi;H;Nakamura;M;Kanamaru;K;Yamagata;H;El-Kady;WA;Kaminishi Y;Itakura;T
• 通讯作者: T

Determination of phosphorylated amino acid residues of Rab8 from Bombyx mori

家蚕 Rab8 磷酸化氨基酸残基的测定

• 发表时间: 2007
• 期刊: Arch. Insect Biochem. Physiol 66
• 作者: Uno;T.;Nakada;T.;Okamaoto;S.;Nakamura;M.;Matsubara;M.;Imaishi;H.;Yamagata;H.;Kanamaru;K. and Takagi;M.
• 通讯作者: M.

Bioconversion by functional P450 1A9 and P450 1C1 of Anguilla japonica

日本鳗鲡功能性 P450 1A9 和 P450 1C1 的生物转化

• 发表时间: 2007
• 期刊: Comprehensive biochemistry and physiology, partC 147
• 作者: Tsukada;M.;D. Tanaka and H. Higuchi;塚田森生・乾未来・須崎徳高・米本仁巳・樋口浩和;塚田 森生・乾 未来・須崎 徳高・米本 仁巳・樋口 浩和;宇野 知秀
• 通讯作者: 宇野 知秀

Monoclonal antibody against Rab8 from Bombyx mori

家蚕 Rab8 单克隆抗体

• 发表时间: 2007
• 期刊: European journal of entomology (印刷中)
• 作者: Uno;T;Okamoto;S;Masuda;S;Imaisihi;H;Nakamura;M;Kanamaru;K;Yamagata;H;El-Kady;WA;Kaminishi Y;Itakura;T;宇野 知秀;宇野 知秀
• 通讯作者: 宇野 知秀