STUDIES ON THE STRUCTURE AND FUNCTION OF THE GLYCINE CLEAVAGE SYSTEM

1.从重叠的克隆中获得了人甘氨酸裂解系统T蛋白的全长cDNA序列。1209个碱基对的开放阅读框编码403个氨基酸的前体蛋白，包括27个残基的线粒体前序列和375个残基的成熟蛋白。该成熟蛋白的氨基酸序列与牛、鸡和大肠杆菌T蛋白的同源性分别为90%、68%和28%。4个物种之间的保守区有限。2.从人胎盘粘粒文库中克隆了人T蛋白基因。该基因全长约6kb，有9个外显子，经荧光原位杂交鉴定为3p21.2-p21.1亚带。3.在分子水平上对两个T-酪氨酸氨基转移酶缺乏所致的非酮症高血糖家系进行了研究，发现了导致Gly19Arg、Gly241Asp和Arg292His氨基酸替换的三个不同点突变。Gly241在不同种属…的T蛋白中保守在大肠杆菌中，Gly19和Arg292分别被Ala和Leu取代。缺失N-端16个氨基酸的T-蛋白在大肠杆菌中表达时，没有显示T-蛋白的活性。为了研究N-末端的作用，我们对N-末端截短不同的E.coliT蛋白进行了过表达。缺少4个残基的T-蛋白的比活力是全长T-蛋白的20%。当缺失7个或更多残基时，比活力约为ET的5%。对缺少7个氨基酸残基的T蛋白(ETDELTA7)进行了纯化，并与ETDELTA7的CD光谱和动力学特性进行了比较。两种蛋白质的圆二色谱无显著差异。但ETDELTA7对大肠杆菌H蛋白的亲和力显著降低。这些结果表明，T蛋白的N端氨基酸序列在与H蛋白的相互作用中起着重要作用。较少

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