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

19 d -氨基酸氧化酶（DAO）反应产生的d -丙基甘氨酸氧化产物抑制了(14C) -< α >-甲基葡萄糖苷进入猪肾近端小管细胞系LLC-PK_1细胞的摄取。抑制程度为正常摄取的95%。由于分离后产物不稳定，无法确定其化学结构。然而，通过核磁共振波谱分析表明，该物质是2-氨基-4-羟基-2,4-戊二酸酯-< γ >-内酯。本文设计了一种核磁共振测量装置，用于测量离子在硝酸纤维素过滤器上通过单层lc - pk1细胞的传输，并测量了Na^+的传输。通过与合成的寡核苷酸杂交，从猪肾cDNA文库中分离到编码DAO的互补dna，并对其核苷酸序列进行了测定。利用分离的克隆建立了体外DAO合成体系，并通过定点诱变对克隆的单核苷酸进行更多的替换，建立了突变体DAO的体外合成体系。结果表明，均经d -丙基甘氨酸修饰的Tyr（228）和His（307）对于维持DAO活性至关重要。dy -品系小鼠肾脏匀浆具有不同的DAO活性。southern和northern blot分析表明，DAO活性的差异引起了DAO基因表达的转录控制。本文以胰蛋白酶为模型激活剂，研究了人尿原allidrein的激活机制。原激肽因与胰蛋白酶反应而迅速产生激肽活性的原因是原激肽释放出前肽，而快速活化后激肽活性的缓慢增加是由于单链激肽转化为双链激肽所致。完整的氨基酸序列已确定。因此，再次证明了快速激活是由于Arb(-1) -Ile(1)键的断裂，而缓激肽活性的缓慢增加是由于Arg(87)-Gln（88）键的断裂。氨基酸序列表明Asn（78）、（84）和（141）存在寡糖结合位点。原激肽激酶与激肽激酶的免疫化学性质不同，尽管两种激肽激酶的分子结构差异不大。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