1992 Fiscal Year Final Research Report Summary
Molecular biologic studies on HHH syndrome
| Project/Area Number |
02454247
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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 |
Neurology
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| Research Institution | KYUSYU UNIVERSITY |
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Principal Investigator |
SUZUKI Tomokazu Kyusyu University, Medical Institute of Bioregulation, Clinical Genetics, Professor, 生体防御医学研究所, 教授 (20028517)
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| Co-Investigator(Kenkyū-buntansha) |
ABE Masako Kyusyu University, Medical Institute of Bioregulation, Clinical Genetics, Resear, 生体防御医学研究所, 助手 (30222665)
MASHIMO Masami Kyusyu University, Medical Institute of Bioregulation, Clinical Genetics, Resear, 生体防御医学研究所, 助手 (80159144)
SUZUKI Yasuyo Kyusyu University, Medical Institute of Bioregulation, Clinical Genetics, Resear, 生体防御医学研究所, 助手 (90226564)
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| Project Period (FY) |
1990 – 1992
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| Keywords | HHH syndrome / Hyperammonemia / Urea cycle / Hyperornithinemia / Inborn errors of metabolism / Mitochondria / Ornithine transport / Affinity labeling |
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| Research Abstract |
HHH syndrome (Hyperammonemia, Hyperornithinemia, and Homocitrullinuria) is an autosomal recessive disorder. It is considered that this syndrome is caused by a defect in the import of ornithine into the liver mitochondria. To elucidate the etiology of this syndrome, we intended to purify the ornithine translocator by the affinity labeling. The screening of the inhibitors by the in vitro citrulline synthesis, showed that the diamine analogs had the inhibitory effects that depended on the distance of nitrogen atoms. Among them, T-46944 inhibited the citrulline synthesis strongly (IC_<50>=0.3mM) and had no effect on other enzymes of citrulline synthesis in mitochondria. Also the sulfhydryl reagents inhibited the citrulline synthesis and it is suggested that SH residues are involved in the ornithine translocation. The swelling caused by the influx of ornithine into mitochondria needed the existence of respiratory substance and phosphate. The swelling was inhibited by T-46944, P-chloromercurybenzoate, and praseodymium chloride.
Recently Indiveri et al. reported the purification and characterization of the ornithine translocator. This protein was of M.W. 33,500 and had the same inhibitors as those we found. So we adopted their method to purify the protein. After the blotting of purified protein onto PVDF membrane, the sequence of N-terminal amino acid residues was determined. Based on the sequence "Glu-Ala-Leu-Lys or Thr-Ala-Gln or Glu-Ala-Leu-Lys-Asp-Phe-Asn or Thr", mixed oligonucleotide primers were synthesized for mixed oligonucleotides primed amplification of cDNA using oligo(dT)_<16> or lambda gt10 specific primers as antiprimers. Now we are trying to clone the ornithine translocator cDNA from the rat liver cDNA or cDNA library constructed from lambda gt10.
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Research Products
(12 results)
