Biochemical and Molecular Biological Studies on Factors Related to Kidney Transport and Homeostasis under Normal and Pathological States

正常和病理状态下肾脏转运和稳态相关因素的生化和分子生物学研究

• 批准号: 63570137
• 负责人: MIYAKE Yoshihiro
• 金额: $ 1.34万
• 依托单位: National Cardiovascular Center Research Institute
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63570137/
• 关键词: D-amino acid oxidase; Renin-binding protein; Complementary DNA; Regulation of gene expression; Site-directed mutagenesis; Leucine zipper structure; Recombinant protein expression; Recombinant mutant proteins; 相補的DNA塩基配列; 遺伝子発現; 翻訳調節; 変異体蛋白質精製 /

1. A complementary DNA (cDNA) encoding human kidney D-amino acid oxidase (DAO) was isolated from a human kidney cDNA library, and the nucleotide sequence was determined.2. A recombinant porcine DAO Was expressed in a large scale in. The expressed recombinant DAO was purified from the cell extract, and the molecular and enzymological properties were characterized.3. Two kinds of recombinant mutant DAOs, in which Tyr-228 or His-307 is replaced with Phe or Leu, respectively, were expressed in Escherichia coli cells. The mutant DAOs were purified from the cell extract, and the functional abnormalities were shown.4. A cDNA encoding rabbit kidney DAO was isolated from a rabbit kidney cDNA library, and the nucleotide sequence was determined. Moreover, the presence of translational suppression in the process of rabbit DAO gene expression was found.5. A cDNA encoding mouse kidney DAO was isolated from a mouse kidney cDNA library, and the nucleotide sequence was determined.6. cDNAs encoding porcine, human and rat renin-binding proteins (RnBP) were isolated from cDNA libraries and the nucleotide sequences were determined.7. It was shown that RnBP is a new protein that has not been identified and it contains a new type of leucine zipper structure.8. The leucine zipper motif in RnBP molecule was shown to participate in the protein function.

1．从人肾cDNA文库中分离编码人肾D-氨基酸氧化酶(DAO)的互补DNA(cDNA)，并测定其核苷酸序列。 2．大规模表达重组猪DAO。从细胞提取物中纯化表达的重组DAO，并对其分子和酶学性质进行表征。 3．在大肠杆菌细胞中表达了两种重组突变DAO，其中Tyr-228或His-307分别被Phe或Leu取代。从细胞提取物中纯化出突变型DAOs，并显示其功能异常。 4．从兔肾cDNA文库中分离编码兔肾DAO的cDNA，并测定其核苷酸序列。此外，还发现兔DAO基因表达过程中存在翻译抑制现象。 5．从小鼠肾脏cDNA文库中分离得到编码小鼠肾脏DAO的cDNA，并测定其核苷酸序列。 6．从cDNA文库中分离编码猪、人、大鼠肾素结合蛋白(RnBP)的cDNA，并测定其核苷酸序列。 7．结果表明，RnBP是一种尚未鉴定的新蛋白，含有一种新型的亮氨酸拉链结构。 8． RnBP 分子中的亮氨酸拉链基序被证明参与蛋白质功能。

项目成果

Motoshige Miyano,Kiyochi Fukui,Fusao Watanabe,Saori Takahashi,Masazumi Tada,Masaru Kanashiro,Yoshihiro Miyake: "Studies on Phe 228 and Leuー307 recombinant mutants of porcine kidney Dーamino acid oxidase:expression,purification,and characterization" The Jou

Motoshige Miyano、Kiyochi Fukui、Fusao Watanabe、Saori Takahashi、Masazumi Tada、Masaru Kanashiro、Yoshihiro Miyake：“猪肾 D 氨基酸氧化酶的 Phe 228 和 Leu-307 重组突变体的研究：表达、纯化和表征” The Jou

Eds.K.Abe,et al.,Saori Takahashi,Akiko Irie,Yoshiaki Katayama,Yoshihiro Miyake (分担執筆): "KININS V,Part A" Plenum Press,New York, 662 (1989)

Eds.K.Abe 等人、Saori Takahashi、Akiko Irie、Yoshiaki Katayama、Yoshihiro Miyake（撰稿人）：“KININS V，A 部分”Plenum Press，纽约，662 (1989)

Kyoko Momoi, Kiyoshi Fukui, Fusao Watanabe, and Yoshihiro Miyake: "Molecular cloning and sequence analysis of cDNA encoding human kidney D-amino acid oxidase" FEBS Lett.238(1). 180-184 (1988)

Kyoko Momoi、Kiyoshi Fukui、Fusao Watanabe 和 Yoshihiro Miyake：“编码人肾 D-氨基酸氧化酶的 cDNA 的分子克隆和序列分析”FEBS Lett.238(1)。

Fukui Kiyoshi, Kyoko Momoi, Fusao Watanabe, and Yoshihiro Miyake: "In vivo and in vitro expression of porcine D-amino acid oxidase ; in vitro system for the synthesis of a functional enxyme" Biochemistry. 27(18). 6693-6697 (1988)

Fukui Kiyoshi、Kyoko Momoi、Fusao Watanabe 和 Yoshihiro Miyake：“猪 D-氨基酸氧化酶的体内和体外表达；用于合成功能性酶的体外系统”生物化学。

Hiroyasu Inoue, Kiyoshi Fukui, Saori Takahashi, and Yoshihiro Miyake: "Molecular cloning and sequence analysis of a cDNA encoding a porcine kidney renin-binding protein" J. Biol. Chem.265(12). 6556-6561 (1990)

Hiroyasu Inoue、Kiyoshi Fukui、Saori Takahashi 和 Yoshihiro Miyake：“编码猪肾肾素结合蛋白的 cDNA 的分子克隆和序列分析” J. Biol。