• Search Research Projects
  • Search Researchers
  • How to Use
  1. Back to previous page

Studies on Carboxyl Proteinases : Comparative Analysis of Functional Structures and Catalytic Mechanisms of Proctases A and B

Research Project

Project/Area Number 05453209
Research Category

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

Allocation TypeSingle-year Grants
Research Field Structural biochemistry
Research InstitutionThe University of Tokyo

Principal Investigator

TAKAHASHI Kenji  The University of Tokyo Graduate School of Science Professor, 大学院・理学系研究科, 教授 (70011533)

Co-Investigator(Kenkyū-buntansha) MURAMATSU Tomonari  The University of Tokyo Graduate School of Science Assistant, 大学院・理学系研究科, 助手 (70212256)
INOUE Hideshi  The University of Tokyo Graduate School of Science Assistant, 大学院・理学系研究科, 助手 (20184765)
TANOKURA Masaru  The University of Tokyo Biotechnology Research Center Professor, 生物生産工学センター, 教授 (60136786)
Project Period (FY) 1993 – 1994
Project Status Completed (Fiscal Year 1994)
Budget Amount *help
¥7,500,000 (Direct Cost: ¥7,500,000)
Fiscal Year 1994: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1993: ¥6,000,000 (Direct Cost: ¥6,000,000)
KeywordsProctase A / Proctase B / carboxyl proteinase / site-directed mutagenesis / autocatalytic activation / active site asparticacid residue / NMR / X-ray crystallography / カルボキシル プロテアーゼ / 活性中心カルボキシル基 / 前駆体活性化 / 部位指定突然異導入法 / アスパラギン酸プロテアーゼ / 部位指定突然変異導入法
Research Abstract

1.By specific chemical modifications, carboxyl groups were shoun to be involved in the active site of Proctase A as in the case of Proctase B.Asp14 and Asp 111 in the heavyc hain of Proctase A were indicated to be the catalytic groups by site-directed mutagenesis studies using an E.coli expression system for pro-Proctase A.
2.A cDNA of prepro-Proctase B was cloned and sequenced, giving the complete amino acid sequence of the preproenzyme. Pro-Proctase B was shown to be autocatalytically activated under acidic conditions. Site-direcited mutagenesis studies on pro-Proctase B revealed that Lys 36 and Arg 32 are important for the shroctural stability of the proform and its activation, respectively.
3.The expression system of pro-Proctase A in E.coli was established, and the proenzyme was shown to be autocatalytically activated under acidic conditions like pro-Proctase B.
4.The profiles of denaturation of Proctase A at different pHs and temperatures were in vestigated using various physico-chemical methods in clading NMR and circular dichroism, which revealed unique denaturation profile of the enzyine.
5.Nearly all proton signals in NMR of the light chain of Proctase A were assigned and the three-dcmensional structure of the chain was determined. Further, a method for preparation of isotopically labeled Proctase A using Aspergillus niger was established, and using these ^<15>N and/or ^<13>C-labeled Protase A,the three-dimensional structure was investigated.
6.A unique substrate specificity of Proctase A was elucidatid using oxidized B chain of insulin ets, which in as quite differant from that of Proctase B.In addition, the interaction of an inhibitor peptide with Proctase A was investigated by NMR.
7.Isomorphous metal (Pt and Hg)derivatives of Proctase A were crystallized and the three-dimensional shructure of the enzyrne was partly elarified by X-ray crystallographic analysis.

Report

(3 results)
  • 1994 Annual Research Report   Final Research Report Summary
  • 1993 Annual Research Report
  • Research Products

    (32 results)

All Other

All Publications (32 results)

  • [Publications] Masaru Tanokura: "A New Crystal Form of Proteinase A,a Non-Pepsin-Type Acid Proteinase from Aspergillus niger var.macrosporus" Journal of Biochemistry. 114. 457-458 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Masaru Tanokura: "Crystallographic Study of Proteinase A,a Non-Pepsin-Type Acid Proteinase from Aspergillus niger var.macrosporus" Photon Factory Report. 11. 110-110 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Hideshi Inoue: "Aspergillus niger var.macrosporus Proteinase B.cDNA Cloning,Expression,Folding and Autoprocessing of the Proenzyme" Advances in Experimental Medicine and Biology. 362(in press). (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Takaaki Hayashi: "Expression and Secretion of Recombinant Aspartic Proteinases by Bacillus brevis" Advances in Experimental Medicine and Biology. 362(in press). (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Naofumi Kagami: "Expression in E.coli of Aspergillus niger var.macrosporus,a Non-Pepsin-Type Acid Proteinase" Advances in Experimental Medicine and Biology. 362(in press). (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Hiroshi Sasaki: "X-Ray Crystallographic Study of a Non-Pepsin-Type Acid Proteinase,Aspergillus niger Proteinase A" Advances in Experimental Medicine and Biology. 362(in press). (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Masaki Kojima: "Conformation Analysis of Non-Pepsin-Type Acid Proteinase from the Fungus Aspergillus niger by NMR" Advances in Experimental Medicine and Biology. 362(in press). (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Jing-Fang Lu: "Molecular Cloning of a cDNA for Proctaase B from Aspergillus niger var.macrosporus and Sequence Comparison with Other Aspergillopepsins I" Bioscience,Biotechnology and Biochemistry. 59(in press). (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Kenji Takahashi: "Proteinase A(Aspergillus niger)" Methods in Enzymology. 248. 146-155 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Harumi Fukuda: "Differential Scanning Calorimetric Studies of the Thermal Unfolding of Acid Proteinase A from Aspergillus niger at Various pHs" Thermochimica Acta. (in press). (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Masaru Tanokura, Hiroshi Sasaki, Tomonari Muramatsu, So Iwata, Toru Hamaya, Toshio Takizawa and Kenji Takahashi: ""A New Crystal Form of Proteinase A,a Non-Pepsin-Type Acid Proteinase from Aspergillus niger var.macrosporus"" J.Biochem.114. 457-458 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Masaru Tanokura, Hiroshi Sasaki, Tomonari Muramatsu, Atsushi Nakagawa and Kenji Takahashi: ""Crystallographic Study of Proteinase A,a None-Pepsin-Type Acid Proteinase from Asperillus niger var.macrosporus"" Photon Factory Report. 11. 110-110 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Hideshi Inoue, Jing-Fang Lu, Senarath B.P.Athauda, Kwang-Hoon Kong, Takaharu Hayashi, Takao Kimura, Osamu Makabe and Kenji Takahashi: ""Aspergillus niger var.macrosporus Proteinase B.cDNA Cloning, Expression, and Activation of the Proenzyme"" Adv.Exp.Med.Biol.362 (in press). (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Takaharu Hayashi, Hideshi Inoue, Masashi Kato, Shigezo Udaka and Kenji Takahashi: ""Expression and Secretion of Recombinant Aspartic Proteinases by Bacillus brevis"" Adv.Exp.Med.Biol.362 (in press). (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Naofumi Kagami, Hideshi Inoue, Takao Kimura, Osamu Makabe and Kenji Takahashi: ""Expression in E.coli of Aspergillus niger var.macrosporus Proteinase A,a Non-Pepsin Type Acid Proteinase"" Adv.Exp.Med.Biol.362 (in press). (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Hiroshi Sasaki, Masaru Tanokura, Tomonari Muramatsu, Atsushi Nakagawa, So Iwata, Toru Hamaya, Toshio Takizawa, Toshiaki Kono and Kenji Takahashi: ""X-Ray Crystallographic Study of a Non-Pepsin-Type Acid Proteinas Aspergillus niger Proteinase A"" Adv.Exp.Med.Biol.362 (in press). (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Masaki Kojima, Masaru Tanokura, Yutaka Muto, Hiroshi Miyano, Ei-ichiro Suzuki, Toru Hamaya, Toshio Takizawa, Toshiaki Kono and Kenji Takahashi: ""Conformation Analysis of Non-Pepsin-Type Acid Protienase from the Fungus Aspergillus niger by NMR"" Adv.Exp.Med.Biol.362 (in press). (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Jing-Fang Lu, Hideshi Inoue, Takao Kimura, Osamu Makabe and Kenji Takahashi: ""Molecular Cloning of a cDNA from Aspergillus niger var.macrosporus and Sequence Comparison with other Aspergillopepsins I."" Biosci, Biotech.Biochem.59 (in press). (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Kenji Takahashi: ""Proteinase A (Aspergillus niger)"" Methods in Enzymology, Proteolytic Enzymes, Part E : Aspartic, Metallo and Other Peptidases (A,J.Barrett, ed.)Academic Press, Orlando. 248. 146-155 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Harumi Fukada, Katsutada Takahashi, Michio Sorai, Masaki Kojima, Masaru Tanokura and Kenji Takahashi: ""Differential Scanning Calorimetric Studies on the Thermal Unfolding of Acid Proteinase A from Aspergillus niger at Various pHs"" Thermochim.Acta. (in press). (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Takashi Hamabata: "Purification,Characterization,and Localization of Follipsin,a Novel Serine Proteinase from the Fluid of Porcine Ovarian Follicles" Journal of Biological Chemistry. 269. 17899-17904 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Masashi Matsushima: "Structural Characterization of Porcine Enteropeptidase" Journal of Biological Chemistry. 269. 19976-19982 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Masaki Kojima: "Thermal Stabilization of Ribonuclease T_1 by Carboxymethylationat Glu-58 as Revealed by ^1H Nuclear Magnetic Resonance Spectroscopy" FEBS Letters. 351. 389-392 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Yuichi Tsuchiya: "Purification and Characterization of a Novel Membrane-Bound Arginine-Specific Serine Proteinase from Porcine Intestinal Mucosa" Jaurnal of Biological Chemistry. 269. 32985-32991 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Yong-Tae Kim: "Leader Peptidase from Escherichia coli.Overexpression,Chracterization and Inactivation by Modification of Tryptophan Residues 300 and 310 with N-Bromosuccinimide" Journal of Biochemistry. 117(in press). (1995)

    • Related Report
      1994 Annual Research Report
  • [Publications] Gwang-Ho Jeohn: "Purification and Characterization of a Vasoactive Intestinal Polypeptide-Degrading Endoprotease from Porcine Antral Mucosal Membranes" Journal of Biological Chemistry. 270(in press). (1995)

    • Related Report
      1994 Annual Research Report
  • [Publications] Takashi Uchino: "Isolation and Characterization of a Novel Serine Proteinase Complexed with α_2-Macroglobulin from Porcine Gastric Mucosa" Journal of Biological Chemistry. 268. 527-533 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Takayuki Takahashi: "Cleavage Specificity and Inhibition Profile of Proteasome Isolated from the Cytosol of Xenopus Oocyte" Journal of Biochemistry. 113. 225-228 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Yoshiaki Tamanoue: "Purification and Characterization of Two Isoforms of Serine Proteinase from the Microsomal Membranes of Rat Liver" Journal of Biochemistry. 113. 229-235 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Senarath B.P.Athauda: "Inhibition of Cathepsin E by α_2-Macroglobulin and the Resulting Structural Changes in the Inhibitor" Journal of Biochemistry. 113. 526-530 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Senarath B.P.Athauda: "Entrapment and Inhibition of Human Immunodeficiency Virus Proteinase by α_2-Macroglobulin and Structural Changes in the Inhibitor" Journal of Biochemistry. 113. 742-746 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Masaru Tanokura: "A New Crystal Form of Proteinase A,a Non-Pepsin-Type Acid Proteinase from Aspergillus niger var macrosporus" Journal of Biochemistry. 114. 457-458 (1993)

    • Related Report
      1993 Annual Research Report

URL: 

Published: 1993-04-01   Modified: 2016-04-21  

Information User Guide FAQ News Terms of Use Attribution of KAKENHI

Powered by NII kakenhi