| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1994: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1993: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
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
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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
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