1987 Fiscal Year Final Research Report Summary
Cloning and expression of dihydrolipoamide acyl-transferase gene and localization of its product in mitochondria
| Project/Area Number |
60480503
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
代謝生物化学
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| Research Institution | Nagasaki University |
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Principal Investigator |
KOIKE Masahiko Nagasaki University School of Medicine Professor, 医学部, 教授 (10039521)
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| Co-Investigator(Kenkyū-buntansha) |
YANAGITA Yutaka Nagasaki University School of Nedicine Instructor, 医学部, 助手 (80191162)
URATA Yoshishige Nagasaki University School of Medicine Instructor, 医学部, 助手 (30185087)
KOIKE Kichiko Nagasaki University School of Medicine Associate Professor, 医学部, 助教授 (80039619)
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| Project Period (FY) |
1985 – 1987
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| Keywords | Dihydrolipoamide Acyl-transferase / mRNA / cDNA / Cloning / Nucleotide Sequence / Amino Acid Sequences / 前駆体の局在化 |
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| Research Abstract |
1. Porcine heart dihydrolipoamide acetyl- and succinyl-transferase(LAT, LST), which are a core enzyme of pyruvate and 2-oxoglutarate dehydrogenase complexes (PDC, OgDC), respectively, were isolated from PDC and OGDC by a single HPLC, and contained covalently bound-lipoic acid as prosthetic group. Both enzymes did not exhibit organ organ specificities.
2. Antisera against two enzymes were raised in rabbit and showed high cross-reactivities with human enzymes. Intra-mitochondrial distribution of LST was examined by an immunocytochemical technique and the gold particles representing LST were found in the matrix along crista.
3. Poly (A)^+ RNAs were isolated from a day-old porcine heart by guanidine isothiocyanate extraction. The precursors of LAT (80KDa) and LST (50KDa) were detected by immunoprecipitation from an ^<35>S-labeled cell-free translation products. The cDNA (600bp - 2kb) library in <lambda>gtll vector were prepared. Transport of the two precursors into rat mitochondria were atte
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mpted.
4. Human LAT and LST cDNA fragments were isolated from HeLa cell <lambda>gtll cDNA library by immunoscreening and the almost full-length cDNAs were isolated from human foreskin fibroblast plasmid library by colony hybridization with phage cDNAs. Nucleotide sequence analyses of plasmid clones revealed an insert of 2.3kb for LAT and one of 2.9kb for LST, respectively. From human placenta cDNA library positive phage clones were isolated and sequence analyses revealed an insert of 1.9kb for LAT and one of 2.8kb for LST, respectively.
5. Mino acid sequences of NH_2-termini of porcine LAT (38 residues) and LST (32 residues) and those of COOH-termini of Two enzymes (5 residues) were determined and compared with those of human enzymes deduced from the nucleotide sequences. The homologies of the predicted sequences of two enzymes among two spacies were poor.
6. By using cloned cDNAs for human LAT and LST the screening of genomic dNAS for two enzymes are attempled from human leucocyte genomic DNA library in EMBL4. Less
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