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

19 D-氨基酸氧化酶(DAO)氧化产物D-丙叉甘氨酸对猪肾小管上皮细胞系LLC-PK_1摄取~(14)C-α&GT；-甲基葡萄糖苷有抑制作用，抑制程度为正常摄取的95%。由于分离不稳定，无法确定产物的化学结构。经1H-核磁共振波谱分析表明，该产物为2-amino-4-hydroxy-2，4-pentadienoate-&lt；gamma&gt；。2.设计了一种测定硝酸纤维素膜上单层LLC-PK1细胞离子转运的核磁共振装置，并测定了Na~+的转运。3.通过与人工合成的寡核苷酸杂交，从猪肾cDNA文库中分离出编码DAO的DNA，并测定了其核苷酸序列。利用分离克隆建立了DAO体外合成体系，并在…后建立了突变型DAO体外合成体系结果表明，经D-丙叉甘氨酸修饰的Tyr(228)和His(307)是维持DAO活性所必需的。Ddy系小鼠肾脏匀浆表现出不同的DAO活性。Southern和Northern印迹分析表明，DAO活性的差异导致了DAO基因表达的转录调控。4.以胰酶为模型激活剂，研究了人尿激肽释放酶的激活机制。激肽释放酶原与胰酶反应迅速出现激肽释放酶活性，快速激活后激肽释放酶活性缓慢增加是由于单链激肽释放酶转化为两链激肽释放酶。结果表明，激肽释放酶的快速激活是由于ARB(-1)-Ile(1)键的断裂，激肽释放酶活性的缓慢增加是由于Arg(87)-Gln(88)键的断裂。从氨基酸序列中可以看出，在Asn(78)、(84)和(141)处存在寡糖结合部位。较少

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