TOMITA Yumiko The Inst. For Enzyme Research, The University of Tokushima, Research Associate, 分子酵素学研究センター, 助手 (00089913)
SAKAI Takashi The Inst. For Enzyme Research, The University of Tokushima, Research Associate, 分子酵素学研究センター, 助手 (80284321)
|Budget Amount *help
¥6,100,000 (Direct Cost : ¥6,100,000)
Fiscal Year 1999 : ¥2,800,000 (Direct Cost : ¥2,800,000)
Fiscal Year 1998 : ¥3,300,000 (Direct Cost : ¥3,300,000)
D-amino acid oxidase (DAO) is a flavoenzyme with FAD as its prosthetic group that catalyzes the oxidative deamination of wide range of D-amino acids. In mammals, DAO is found in highest concentrations in kidney, liver and brain. Recent reports that D-serine is present is brain and the distribution of DAO in the brain is inversely correlated to that of D-serine, prompted us to investigate the functional correlation of DAO with D-serine in brain.
In search of nervous system specific expression of DAO as the first step, we have cloned the rat cerebellar cDNA and predicted the primary structure of brain DAO. Analysis of the nucleotide sequence (nt) revealed that full length cDNA has a 1547 nt sequence with a 5'-untranslated region of 199 nt, an open reading frame of 1041 nt, and 3'-untranslated region of 307 nt that contains the polyadenylation signal sequence. The deduced amino acid sequence consisting of 346 amino acids showed 93.1, 80.7, 77.8 and 79.0% identity with the mouse, human, por
cine and rabbit kidney enzyme, respectively. Three catalytically important residuces, Tyr-224, Tyr-228 and Arg-283, of the porcine enzyme were all conserved in these 4 species. The targeting signal for the peroxisome localization of the brain enzyme was also present at the C-terminal sequence, Ser-His-Leu. Then we have established the culture systems of cerebral and cerebellar astrocytes and detected the gene expression of DAO in type 1 astrocyte. Moreover, the effect of D-serine on the cultured astrocytes was examined to investigate the induction of the gene expression of DAO and the cell death.
Renin binding protein (RnBP) is a protein that binds to renin to form a protein complex called high molecular weight (HMW) renin. This protein inhibits renin activity. In search of its physiological function, gene expression of RnBP in rat aorta were examined by Northern blot hybridization. RnBP gene expression was observed in rat aorta, in addition to kidney, adrenal gland, brain and ovary.
Using In Situ RT-PCR analysis to determine RnBP mRNA localization in rat kidney, it was suggested that RnBP mRNA was expressed in juxtamedullary glomerulus and vascular endothelial cells in glomerulus. The arterial endothelial cells derived from porcine aorta exhibited high level of RnBP gene expression, detected by Northern blot hybridization analysis. In order to study renin gene expression in relation to that of RnBP, porcine renin cDNA fragment was isolated by RT-PCR analysis of porcine kidney mRNA, followed by determination of the nucleotide sequence for porcine renin. Finally, RT-PCR analysis of porcine renin mRNA in endothelial cells has shown that renin gene is also expressed in the same endothelium. RnBP gene expression was shown to be upregulated by Angiotensin II and TGF-data, but noto by TNF-alfa in cultured endothelial cells. Therefore, RnBP regulation by angiotensin II sugggests that RnBP participate in blood pressure regulation at vascular endothelum as a member of vascular renin-angiotensin system. Less