| Project/Area Number |
02454266
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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 |
Pediatrics
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| Research Institution | Tohoku University |
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Principal Investigator |
NARISAWA Kuniaki Tohoku University, School of Medicine, Professor, 医学部, 教授 (90004647)
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| Co-Investigator(Kenkyū-buntansha) |
SUZUKI Youichi Tohoku University, School of Medicine, Research Associate, 医学部, 助手 (80216457)
MATSUBARA Youichi Tohoku University, School of Medicine, Associate Proffessor, 医学部, 助教授 (00209602)
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| Project Period (FY) |
1990 – 1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥6,800,000 (Direct Cost: ¥6,800,000)
Fiscal Year 1991: ¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1990: ¥4,600,000 (Direct Cost: ¥4,600,000)
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| Keywords | multiple carboxylase deficiency / biotin dependency / holocarboxylase synthetase / enzyme purification / enzyme diagnosis |
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| Research Abstract |
Neonatal multiple carboxylase deficiency presents as life-threatening acidotic illness in the earliest days of life. We have shown to be due to a defect in the enzyme holocarboxylase synthetase (HCS) which is essential for the attachment of biotin to the inactive apocarboxylase enzymes. Fibroblasts from a patient were found to have deficient activities of PCC, MCC, PC and ACC and have abnormal HCS activity with a highly elevated Km for biotin. HCS has been purified in nearly homogeneous form from bovine liver cytosol by the sequence of ammonium sulfate fractionation, Almina Cr fractionation, DEAE-SepharoseCL-6B, EAH-Sepharose 4B, Sephacryl S-200 HR, Hydroxyapatite HTP and Phenyl-Superose HR 5/5 chromatographies. A novel HCS assay method was adopted for this study utilizing propionly-CoA apocarboxylase from cultured lymphoblasts of HCS deficient patient as the substrate. The purified enzyme showed a single protein band on SDS PAGE with a molecular weight of 64, 000. HCS is a monometric protein. Its apparent Km values were 58 nM for biotin and 28.6 mu M for ATP. Tryptic digests of HCS, reverse-phase. HPLC separations of tryptic peptides, and amino acid analyses of four of the separated peptides were performed. A cDNA coding for the HCS was cloned from a bovine liver cDNA library by screening with synthetic oligonucleotide probes.
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