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Studies on the molecular properties and the structure-function relationships of carboxyl proteases of novel type

Research Project

Project/Area Number 02453150
Research Category

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

Allocation TypeSingle-year Grants
Research Field 物質生物化学
Research InstitutionThe University of Tokyo

Principal Investigator

TAKAHASHI Kenji  Univ. of Tokyo, Faculty of Science, Professor, 理学部, 教授 (70011533)

Co-Investigator(Kenkyū-buntansha) INOUE Hideshi  Univ. of Tokyo, Faculty of Science, Assistant, 理学部, 助手 (20184765)
TAKAHASHI Takayuki  Univ. of Tokyo, Faculty of Science, Lecturer, 理学部, 講師 (80197152)
TANOKURA Masaru  Univ. of Tokyo, Faculty of Science, Lecturer, 理学部, 講師 (60136786)
丹治 雅夫  東京大学, 理学部, 特別研究員(PD) (50212048)
Project Period (FY) 1990 – 1991
Project Status Completed (Fiscal Year 1991)
Budget Amount *help
¥5,800,000 (Direct Cost: ¥5,800,000)
Fiscal Year 1991: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1990: ¥4,400,000 (Direct Cost: ¥4,400,000)
KeywordsCarboxyl protease / Acid protease / Proctase A / Cathepsin E / Copia protease / Physarum acid protease / Protease precursor / Autocatalytic activation / 水溶性カルボジイミド / 基質特異性 / cDNA / X線結晶解析 / NMR / 遺伝子 / トランスポゾンプロテア-ゼ / 粘菌プロテア-ゼ / ペプシノ-ゲン
Research Abstract

1. The complete amino acid sequence of a carbowxl protease of novel type (Proctase A) produced by the fungus, Aspergillus niger var. macrosporus, has been determined by conventional methods of protein chemistry. Further, the gene and cDNA of Proctase A have been cloned and the base sequence of the coding region of the cDNA has been elucidated.
2. The substrate specificity toward various protein and peptide substrates of Proctase A has been elucidated and the active-site residues have been explored by chemical modification. In addition, the cDNA for proproctase A could be expressed in E. coli and shown to be autocatalytically activated under acidic conditions.
3. Proctase A could be crystallized and a preliminary X-ray-study on it has been performed. On the other hand, two-dimensional NMR and circular dichroism studies have revealed that Proctase A has a high beta-sheet structure and a rigid core structure in the molecule.
4. Proctase A has been shown to be denatured rapidly and irreversibly at pH 6-7 with simultaneous dissociation of the two peptide chains.
5. A unique pepstatin-insensitive acid protease (M 54K) has been purified from the plasmodia of a true slime mold, Physarum polycephalum, and shown to have a unique two chain structure and substrate specificity.
6. Procathepsin E has been purified from the gastric mucosa of human stomach, and its NH_2-terminal amino acid sequence and the site of carbohydrate attachment have been clarified. Further, procathepsin E has been shown to be autocatalytically activated and to have significant proteolytic activity at neutral pH with rather narrower substrate specificity.
7. The gene for Drosophila copia protease could be expressed in E. coli, giving an active enzyme. Its cleavage specificity toward the precursor protein has been elucidated, and the importance for activity of an aspartic acid residue at the putative active site has been demonstrated by site-directed mutagenesis.

Report

(3 results)
  • 1991 Annual Research Report   Final Research Report Summary
  • 1990 Annual Research Report
  • Research Products

    (32 results)

All Other

All Publications (32 results)

  • [Publications] S.B.P.Athauda: "Structural evidence for two isozymic forms and the carbohydrate attachment site of human gastric cathepsin E" Biochemical and Biophysical Research Communications. 168. 878-885 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K.Murakami-Murofushi: "Purification and characterization of a novel intracellular acie proteinase from the plasmodia of a true slime mold,Physarum polycephalum" Journal of Biological Chemistry. 265. 19898-19903 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] S.B.P.Athauda: "Autocatalytic processing of procathepsin E to cathepsin E and their structural differences" Biochemical Biophysical Research Communications. 175. 152-158 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K.Takahashi: "The Primary Structure of Aspergillus niger Acid Proteinase A" Journal of Biological Chemistry. 266. 19480-19483 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] H.Inoue: "The Gene and Deduced Protein Sequences of the Zymogen of Aspergillus niger Acid Proteinase A" Journal of Biological Chemistry. 266. 19484-19489 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] S.B.P.Athanda: "Proteolytic activity and cleavage specificity of cathepsin E at the physiological pH as examined towards the B enain of oxidized insulin" FEBS Letters. 292. 53-56 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] E.Yakabe: "Purification,Characterization,and Amino Acid Sequences of Pepsinogens and Pepsins from the Esophageal Mucosa of Bullfrog(Rana catesbeiana)" Journal of Biological Chemistry. 266. 22436-22443 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] H.Matsuzaki: "Effect of Dimethylsulfoxide on the Crystallization of Aspergillus niger Proteinase A" Procedings of the Japan Academy. 67. 209-212 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] M.Tanokura: "Crystallization and Preliminary X-ray Investigation of Proteinase A,a Non-pepsin-type Aced Proteinase from Aspergillus niger var,macrospores" Journal of Molecular Biology. 223. 373-375 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K.Takahashi: "Structure and Function of a Pepstatin-Insensitive Acid Proteinase from Aspergillus niger var.Macrosporuo" Advances in Experimental Medicine and Biology. 306. 203-211 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] S. B. P. Athauda: "Structural evidence for two isozymic forms and the carbohydrate attachment site of human gastric cathepsin E" Biochem. Biophys. Res. Commun.168. 878-885 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K. Murakami-Murofushi: "Purification and characterization of a novel intracellular acid proteinase from the plasmodia of a true slime mold, Physarum polycephalum" J. Biol. Chem.265. 19898-19903 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] S. B. P. Athauda: "Autocatalytic processing of procathepsin E to cathepsin E and their structural differences" Biochem. Biophys. Res. Commun.175. 152-158 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K. Takahashi: "The Primary Structure of Aspergillus niger Acid Proteinase A" J. Biol. Chem.266. 19480-19483 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] H. Inoue: "The Gene and Deduced Prothein Sequences of the Zymogen of Aspergillus niger Acid Proteinase A" J. Biol. Chem.266. 19484-19489 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] S. B. P. Athauda: "Proteolytic activity and cleavage specificity of cathepsin E at the physiological pH as examined towards the B chain of oxidized insulin" FEBS Lett.292. 53-56 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] E. Yakabe: "Purification, Characterization, and Amino Acid Sequences of Pepsinogens and Pepsins from the Esophageal Mucosa of Bullfrog (Rana catesbeiana)" J. Biol. Chem. 266. 22436-22443 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] H. Matsuzaki: "Effect of Dimethylsulfoxide on the Crystallization of Aspergillus niger Proteinase A" Proc. Japan Acad.67. 209-212 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] M. Tanokura: "Crystallization and Preliminary X-ray Investigation of Proteinase A, a Non-pepsin-type Acid Proteinase from Aspergillus niger var. macrosporus" J. Mol. Biol.223. 373-375 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] K. Takahashi: "Structure and Function of a Pepstatin-Insensitive Acid Proteinase from Aspergillus niger var. Macrosporus" Adv. Exp. Med. Biol.306. 203-211 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] S.B.P.Athauda: "Autocatalytic processing of procathepsin E to cathepsin E and their structural differences" Biochemical Biophysical Research Communications. 175. 152-158 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] K.Takanashi: "The Primary Structure of Aspergillus niger Acid Proteinase A" Journal of Biological Chemistry. 266. 19480-19483 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] H.Inoue: "The Gene and Deduced Protein Sequences of the Zymogen of Aspergillus niger Acid Proteinase A" Journal of Biological Chemistry. 266. 19484-19489 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] S.B.P.Athauda: "Protelytic activity and eleavage specificity of cathepsin E at the physiological pH as examined towards the B chain of oxdized insulin" FEBS Letters. 292. 53-56 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] E.Yakabe: "Purification,Characterization,and Amino Acid Sequences of Pepsinogens and Pepsins from the Esophageal Mucosa of Bullfrog (Rana catesbeiana)" Journal of Biological Chemistry. 266. 22436-22443 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] H.Matsuzaki: "Effect of Dimethylsulfoxide on the crystallization of Aspergillus niger Proteinase A" Proceedings of the Japan Academy. 67. 209-212 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] M.Tanokura: "Crystallization and Preliminary Xーray Investigation of Proteinase A,a Nonーpepsinーtype Acid Proteinase from Aspergillus niger var.macrosporus" Journal of Molecular Biology. 223. 373-375 (1992)

    • Related Report
      1991 Annual Research Report
  • [Publications] K.Takahashi: "Structure and Function of a PepstatinーInsensitive Acid Proteinase from Aspergillus niger var.Macrosporus" Advances in Experimental Medicine and Biology. 306. 203-211 (1992)

    • Related Report
      1991 Annual Research Report
  • [Publications] S.B.P.Athauda: "Structural evidence for two isozymic forms and the carbohydrate attachment site of human gastric cathepsin E" Biochemical and Biophysical Research Communications. 168. 878-885 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] K.Murakami,Murofushi: "Purification and Characterization of a novel intracellular acid proteinase from the plasmodia of a true slime mold,<Physarum>___ー <polycephalum>___ー" Journal of Biological Chemistry. 265. 19898-19903 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] M.Ichinose: "Hydrocortisoneーinduced enhancement of expression and changes in methylation of pepsinogen genes in stomach mucosa of the developing rat" Biochemical Biophysical Research Communications. 172. 1086-1093 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] S.B.P.Athauda: "Autocatalytic processing of procathepsin E to cathepsin E and their structural differences" Biochemical Biophysical Research Communications. (1991)

    • Related Report
      1990 Annual Research Report

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

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