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STUDIES ON THE STRUCTURE AND FUNCTION OF THE GLYCINE CLEAVAGE SYSTEM

甘氨酸裂解系统的结构和功能研究

基本信息

批准号：
05670127
负责人：
OKAMURA Kazuo
金额：
$ 1.34万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

1.The full-length cDNA sequence of human T-protein of the glycine cleavage system was determined from overlapping cDNA clones. The 1209-base pair open reading frame encodes precursor protein of 403 amino acid which consist of 27-residue mitochondrial presequence and 375-residue mature protein. The deduced amino acid sequence of the mature protein shows 90,68 and 28% homology to that of bovine, chicken and Escherichia coli T-protein, respectively. The conserved regions among four species are limited.2.The gene for human T-protein was isolated from a human placental cosmid library. The gene is about 6 kb in length with nine exons and is assigned to subband 3p21.2-p21.1 by fluorescence in situ hybridization.3.Two pedigrees of nonketotic hyperglycinemia caused by T-prorein deficiency have been investgated at molecular level.Three different point mutations which lead to amino acid substitutions Gly19Arg, Gly241Asp and Arg292His were identified. Gly241 is conserved in T-protein of various sp … More ecies, even in E.coli, whereas Gly19 and Arg292 are replaced by Ala and Leu, respectively, in E.coli. Therefore, these substitutions may cause the change in tertiary structure of T-protein.The E.coli T-protein lacking the N-terminal 16 amino acids showed no T-protein ativity, when the truncated T-proein gene was expressed in E.coli. In order to investigate the role of N-terminal region, E.coli T-protein with variously truncated N-terminal portions were overexpressed. The specific activity of T-protein lacking 4 residues are 20% of that of the full length T-protein (ET). The specific activity was about 5% of that of ET when 7 residues or more were deleted. T-protein lacking 7 amino acid residues (ETDELTA7) was purified and its characteristics such as CD spectra and kinetics were compared with ET.ETDELTA7 was eluted at a little higher salt concentration than ET from DEAE-Sepharose column. There is no significant difference in the CD spectra of both proteins. However, the affinity of ETDELTA7 for E.coli H-protein remarkably decreased. These results suggest that the N-terminal amino acid sequence of T-protein seemes to be important for the interaction with H-protein. Less

1.从重叠cDNA克隆中确定了甘氨酸切割系统的人T蛋白的全长cDNA序列。该基因的开放阅读框长1209个碱基，编码403个氨基酸的前体蛋白，由27个氨基酸的线粒体前序列和375个氨基酸的成熟蛋白组成。推导的成熟蛋白氨基酸序列与牛、鸡和大肠杆菌T蛋白的同源性分别为90%、68%和28%。从人胎盘粘粒文库中克隆了人T蛋白基因。该基因长约6 kb，有9个外显子，荧光原位杂交结果显示，该基因位于3p21.2-p21.1亚带。3.从分子水平上对两个由T-蛋白原缺乏引起的非酮症高甘氨酸血症家系进行了研究，发现了3个不同的点突变，分别导致Gly 19 Arg、Gly 241 Asp和Arg 292 His的氨基酸替换。Gly 241在不同物种的T蛋白中是保守的 ...更多信息而Gly 19和Arg 292在大肠杆菌中分别被Ala和Leu取代。因此，这些取代可能会导致T蛋白三级结构的改变，缺失N端16个氨基酸的大肠杆菌T蛋白在大肠杆菌中表达时，不显示T蛋白活性。为了研究N-末端区域的作用，过量表达具有各种截短的N-末端部分的大肠杆菌T-蛋白。缺失4个氨基酸残基的T蛋白比活性约为全长T蛋白的20%。当缺失7个或更多残基时，比活性约为ET的5%。对缺失7个氨基酸残基的T蛋白（ETDELTA 7）进行了纯化，并与ET进行了CD谱和动力学等特性的比较。两种蛋白质的CD光谱无显著差异。然而，ETDELTA 7对大肠杆菌H蛋白的亲和力显著降低。这些结果表明T蛋白N端氨基酸序列对与H蛋白的相互作用具有重要意义。少