| Project/Area Number |
61570143
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Nagasaki University |
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Principal Investigator |
KOIKE Kichiko Nagasaki University School of Medicine ・ Associate Professor, 医学部, 助教授 (80039619)
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| Co-Investigator(Kenkyū-buntansha) |
YANAGITA Yutaka Nagasaki University School of Medicine ・ Instructor, 医学部, 助手 (80191162)
URATA Yoshishige Nagasaki University School of Medicine ・ Instructor, 医学部, 助手 (30185087)
KOIKE Masahiko Nagasaki University School of Medicine ・ Professor, 医学部, 教授 (10039521)
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| Project Period (FY) |
1986 – 1987
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| Project Status |
Completed (Fiscal Year 1987)
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| Budget Amount *help |
¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1987: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1986: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | Thiamin Pyrophosphate-dependent Enzyme / <lambda>gt11 Expression Screening / Colony Hybridization / クローニング / 塩基配列 / アミノ酸配列 / α, βmRNA / α, βゲノムDNA / 【α_2】【β_2】構造 / 攻α,β家兎抗体 / 遺伝子(cDNA) |
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| Research Abstract |
Pyruvate dehydrogenase (PDH) is one of the component enzymes of PDH multienzyme complex, requires thiamin pyrophosphate, and is composed of two 41KDa subunit (PDH<alpha> and two 36KDa subunit (PDH<beta>) in <alpha>_2<beta>_2 form with Mr 153,000.
1. Porcine PDH was isolated from PDH complex by a single HPLC and separated into two subunits by HPLC. Antisera against porcine PDH<alpha> and <beta> were raised in rabbit and cross-reacted with human subunits.
2. Human PDH<alpha> and <beta> cDNA fragments were isolated from a HeLa cell library in <lambda>gt11 vector by immunoscreening and the full-length cDNAs were isolated from human foreskin fibrablast cDNA library by colony hybridization with phage cDNAs.
3. Nucleotide sequence analyses of plasmid clones (pHPDA, pHPDB) revealed an insert of 1.36kb for PDH<alpha> and 1.69kb for PDH<beta>, respectively. A putative leader sequence of 29 amino acid residues was identified in pHPDA, resulting in a precursor of 369 residues (MR 43,414) and a mature protein of 363 residues (Mr 40,334). A similar leader sequence of 30 amino acid residues in pHPDB was also identified resulting in a precursor of 359 residues (Mr 39,046) and a mature protein of 329 residues (Mr 35,911). The amino acid sequences around NH_2-termini of two subunits of the human and porcine PDHs were highly homologous. The sequences around the three phosphorylation sites determined in PDH<alpha> of bovine and porcine enzymes were also conserved.
4. Blot analysis of HeLa cell poly (A)^+ RNA showed single mRNA of 1.8kb for PDH<alpha> and 1.7 kb for PDH<beta>, respectively. The precursor of PDH<alpha> and <beta> were detected by immunoprecipitation from an ^<35>S-labeled cell-free translation system.
5. In a tissue extract with PDH defect new abnomal protein bands were detected by Western blot analysis, suggesting mutation of PDH<alpha> and <bet> genomes. PDH genomic DNA is now under screening from human leucocyte EMBL4 genomic library.
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