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クツラベ病における欠損酵素のクローニング遺伝子異常の解析と遺伝子治療の試み

Research Project

Project/Area Number 02454270
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field Pediatrics
Research InstitutionOsaka University

Principal Investigator

岡田 伸太郎  大阪大学, 医学部, 教授 (30028609)

Co-Investigator(Kenkyū-buntansha) 福島 久雄  大阪大学, 医学部, 助手 (70199214)
乾 幸治  大阪大学, 医学部, 講師 (90175208)
塚本 浩子  大阪大学, 医学部附属病院, 医員
谷池 雅子  大阪大学, 医学部附属病院, 医員
Project Period (FY) 1990 – 1992
Project Status Completed (Fiscal Year 1992)
Budget Amount *help
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1992: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1991: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1990: ¥5,500,000 (Direct Cost: ¥5,500,000)
Keywordsガラクトセレブロシダーゼ / クローニング / クラッベ病 / ガラクトシルセラミダ-ゼ / モノクロ-ナル抗体
Research Abstract

クラッベ病の欠損酵素であるgalactocerebrosiraseのクローニングをめざし、平成3年度に精製酵素を用い常法にてモノクローナル抗体を作成した。得られたLgG1クラスのモノクローナル抗体1-9-1-9にて発現ベクターλgt11のヒト胎盤由来のcDNAライブラリーをスクリーニングしたが有意のpositive cloneを得る事が出来なかった。
そこで精製酵素のアミノ酸配列からオリゴヌクレオナドを合成しこれをブローブとしてcDNAライブラリーのスクリーニングを計画した。精製酵素をSDS-PAGEで分離した分子量約10万のバンドをPVDF膜に転与しN端のアミノ酸配列を気相シークエンサーにて求めたところ最高でアミノ酸14残基まで解続できた。このN端アミノ酸配列より予想されるいくつかのオリゴマーを合成し、これをブローブとして上記のλgt11のcDNAライブラリーをスクリーニング中である。またアミノ酸の内部配列を調べるため、PVDF膜に転写後の蛋白を膜上で酵素消化しペプチドマッピングをとり、得られたペプチドのアミノ酸情報を現在解析中である。
一方精製酵素の生化学的性質について調べたところ、至適pHはlaurocholate系では4.2、Chplate系では4.6であり比活性は各々1.48×10^5nmol/hr/mg、2.30×10^5nmol/hr/mgであった。Lineweaver-BurkフロットよりTaurocholate系でVmax=1.54×10^5nmol/hr/mg、Km=5-10βMともとまった。また本酵素の天然基質とGM1ganglioside、人工蛍光基質6-he|aι|eιano|am|no-4-methy|umbe|lifery|-β-ga|actopvranos|deの阻害実験により後2者は競合阻害を示すことが判明した。

Report

(3 results)
  • 1992 Annual Research Report
  • 1991 Annual Research Report
  • 1990 Annual Research Report
  • Research Products

    (14 results)

All Other

All Publications (14 results)

  • [Publications] Inui K: "Mitochondrial encephalomyopathies with the mutaion of the mitochondrial tRNA Leu(UUR) gene." J Pediatr.120(1). 62-66 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Taniike M: "A case of pigmentary type of orthochromatic leukodystrophy with early onset and globoid cells." Acta neuropathol.83. 427-433 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Taniike M: "Mitochondrial tRNA Ile mutation in fatal cardiomyopathy." Biochem biophys Rse Commun.186(1). 47-53 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Inui K: "Detection of the A to G(3243) mutation of mitochondrial DNA in Japanese families with mitochondrial encephalomyopathies." J Inher Metab Dis.15. 311-314 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Tsukamoto H: "A case of hallervorden-spatz disease:Progressive and intractable dystonia controlled by bilateral thalamotomy." Brain & Development.14. 269-272 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Taniike M,et al.: "Localization of sphingolipid activator proteinー1(SAPー1) in the brain of a normal human and a patient with metachromatic leukodystrophy." Acta Histochem Cytochem. 24(2). 215-222 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Midorikawa M,et al.: "Uptake and metabolism of radiolabelled GM1ーganglioside in skin fibroblasts from controls and patients with GM1ーgangliosidosis." J Inher Metab Dis. 14. 721-729 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Nishimoto J,et al.: "GM1ーgangliosidosis (genetic βーgalactosidase deficiency):identification of four metations in different clinical phenotypes among japanes patients." Am H Hum Genet. 49. 566-574 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Yoda K: "Lysosulfatide(Sulfogalactosyl sphingosine)accumulation in tissues from patients with metachromatic Teulcodystrophy" J.Neurodrem. 55. 1585-1591 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Inui K: "Study of pathogenesis in twitcher mouse,an enzymatically authentic model of Krabbe's clisease" J Neurol Sci. 100. 124-130 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Inui K: "A case of chronic GM_1 gangliosidosis preseriling as dystonia:clinical and biochemical studies" J Neuro'gy. 237. 491-493 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Taniike M: "Ultrasmictural pathology of rectum and skin biopsy specimens in lysosomal storage disease" Acta Histochem Cytochem. 23. 81-92 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Yamashita K: "Characteristics of asparagineーlinked sugar chains of sphingolipid actlvator protein I purified from normal liver asat GM_1 gangliosidosis(type1) liver" BiochemistryーUSA. 29. 3030-3039 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Miclorikawa M: "Uptake and metabolism of rokliolabeled GM_1 ganglioside in skinfibroblasts from confrels and patients with GM_1 gangliocidocis" J Inher Metab Dis.

    • Related Report
      1990 Annual Research Report

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Published: 1990-04-01   Modified: 2016-04-21  

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