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Studies on moiecular and cellular transporting mechanism of nephrons under pathological states

病理状态下肾单位分子和细胞转运机制的研究

基本信息

批准号：
61570151
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：
1986
资助国家：
日本
起止时间：
1986 至 1987
项目状态：
已结题

项目摘要

19Oxidation product of D-propargylglycine from D-amino acid oxidase(DAO) reaction inhibited uptake of (14C) -<Alpha>-methylglucoside into LLC-PK_1 cells,an established cell line from proximal tubules of porcine kidney.The extent of inhibition was 95% of that of normal uptake.The chemical structure of the product could not be detemined owing to unstability after isolation.However,analysis of the material by^1H-NMR spectroscopy suggests that it is 2-amino-4-hydroxy-2,4-pentadienoate-<gamma>-lactone.2.An apparatus for NMR measurement of ion transport across a monolayer of LLC-PK1 cells on a nitrocellulose filter has been contrived,and Na^+ transport was measured.3.Complimentary DNAs encoding DAO have been isolated from porcins kidney cDNA library by hybridization with synthetic oligonucleotides and the nucleotide sequences were determined. In vitro DAO synthesizing system has been established using the isolated clone,and in vitro syntesizing system of mutant DAOs also was established afte … More r replacement of single nucleotide of the clone by site-directed mutagenesis.As the results,Tyr(228) and His(307),both modified by D-propargylglycine,were shown to be essential for essential for maintaining DAO activity. Kidney homogenates from ddY-strain mice exhibited various DAO activity.It is indicated from southern and northern blot analyses that the difference in DAO activity caused transcriptional control of DAO gene expression.4.The activation mechanism of human urinary prokallidrein has been investigated using trypsin as a model activator.The rapid appearance of kallikrein activity by the reaction of prokallikrein with trypsin was due to release of the propeptide from prokallikrein,and the slow increase in kallikrein activity after the rapid activation was due to conversion of the single chain kallikrein to two chain form. The complete amino acid sequence has been determined.As the result,again,it has been demonstrated that the rapid activation is due to cleavage of Arb(-1) -Ile(1) bond and the slow increase in kallikrein activity is due to cleavage of Arg(87)-Gln(88) bond.The presence of oligosaccharide binding site at Asn(78), (84),and (141) was indicated from the amino acid sequence.Immunochemical properties of prokallikrein differed from those of kallikrein,although the difference in molecular structure between the two forms of kallikrein is slight. Less

19. D-氨基酸氧化酶（DAO）反应生成的D-炔丙基甘氨酸氧化产物抑制<Alpha>猪肾近曲小管细胞株LLC-PK_1对（14 C）- -甲基葡萄糖苷的摄取，抑制程度为正常摄取的95%。由于分离后不稳定，产物的化学结构无法确定。但经~ 1H-NMR谱分析表明，它是2-氨基-4-羟基-2，4-异戊烯酸- -β<gamma>-内酯。2.建立了一种NMR测定（14 C）--甲基葡萄糖苷离子的装置用硝酸纤维素膜对LLC-PK 1细胞进行了Na^+跨膜转运实验，并对Na^+跨膜转运进行了测定。利用分离的克隆建立了DAO体外合成体系，并在此基础上建立了突变体DAO体外合成体系。 ...更多信息结果表明，经D-炔丙基甘氨酸修饰的Tyr（228）和His（307）是维持DAO活性所必需的。来自ddY的肾匀浆-4.以胰蛋白酶为模型激活剂，研究了人尿激肽释放酶原的激活机制，发现胰蛋白酶与激肽释放酶原反应后，由于胰蛋白酶释放前肽而迅速产生激肽释放酶的活性，从而使人尿激肽释放酶原的活性迅速增强，并使人尿激肽释放酶原的活性迅速增强。在快速活化后，激肽释放酶活性的缓慢增加是由于单链激肽释放酶转化为双链形式。结果再次证明，激肽释放酶活性的快速激活是由于Arb（-1）-Ile（1）键的断裂，而激肽释放酶活性的缓慢增加是由于Arg（87）-Gln（88）键的断裂。和（141）的氨基酸序列。尽管两种形式的激肽释放酶的分子结构差异很小，但激肽释放酶原的免疫化学性质与激肽释放酶的免疫化学性质不同。少