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

1.从人肾cDNA文库中克隆了编码人肾D-氨基酸氧化酶（DAO）的cDNA，并测定了其核苷酸序列.重组猪DAO在大肠杆菌中得到了大量表达。从细胞提取液中纯化表达的重组DAO，并对其分子和酶学性质进行了表征.将Tyr-228和His-307分别替换为Phe和Leu，在大肠杆菌中表达了两种重组突变型DAO。从细胞提取物中纯化出突变的DAO，并显示出功能异常.从兔肾cDNA文库中克隆了一个编码兔肾DAO的cDNA，并测定了其核苷酸序列。此外，在兔DAO基因的表达过程中存在翻译抑制.从小鼠肾脏cDNA文库中克隆了编码小鼠肾脏DAO的cDNA，并测定了其核苷酸序列.从cDNA文库中分离编码猪、人和大鼠肾素结合蛋白（RnBP）的cDNA，并测定其核苷酸序列.结果表明，RnBP是一种尚未被鉴定的新蛋白，它含有一种新型的亮氨酸拉链结构. RnBP分子中的亮氨酸拉链基序被证明参与蛋白质功能。

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.