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1994 Fiscal Year Final Research Report Summary

ISOLATION AND ANALYSIS OF RECEPTOR FOR INSECTICIDAL PROTEIN, delta-ENDOTOXIN

Research Project

Project/Area Number 04660093
Research Category

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

Allocation TypeSingle-year Grants
Research Field 応用生物化学・栄養化学
Research InstitutionUniversity of Osaka Prefecture, College of Agriculture

Principal Investigator

HIMENO Michio  Univ.of Osaka Pref., College of Agric., Professor, 農学部, 教授 (10026411)

Co-Investigator(Kenkyū-buntansha) SUGIMOTO Kenji  Univ.of Osaka Pref., College of Agric., Assistant, 農学部, 助手 (40196746)
Project Period (FY) 1992 – 1994
KeywordsBiopesticide / Bacillus thuringiensis / Receptor / Mid-gut, silkworm / BBMV / delta-Endotoxin
Research Abstract

The delta-endotoxin produced by Bacillus thuringiensis was known as a crystalline protein body (CPB). This CPB was also used as microbial pesticide. It seem that a factor to determine the specific toxicity of CPB is a receptor in brush border membrane (BBM) of mid-gut epithelial cells. Then in this project, we attempt to isolation and analysis of the receptor from BBM of the silkworm mid-gut for {CryIA(a)}.
An insecticidal protein, CryIA (a) was isolated from E.coli carrying CryIA (a) gene cloned from B.thuringiensis var.aizawai. In order to isolate the receptor for CryIA (a), the brush border membrane vesicle (BBMV) was solubilized by 2% cholic acid, and was adsorbed on the CryIA (a) -immobilized Tresyl Toyopearl affinity column. The eluate from the affinity column was applied on a DEAE-Toyopearl. The binding activities with ^<125>I-CryIA (a) of the purified receptor by the DEAE-column were determined and blocking activities of ^<125>I-CryIA (a) against BBMV or solubilized BBMV were al … More so detected. An electropherogram of the receptor protein on PAGE was determined by the binding activity with ^<125>I-CryIA (a). A main 180 kDa and minor 170 kDa bands were detected on the autoradiogram of ^<125>I.The 170 kDa band was removed by using of proteinase inhibitors in all purification processes. It was confirmed that the only 180 kDa protein was a receptor protein by the competition experiments of ^<125>I-CryIA (a) and BBMV or the solubilized BBMV.The 180 kDa protein was not a leucine aminopeptidase or an alkaline phosphatase. It was determined that the molecular weight of the native receptor protein was about 600 kDa by a gel filtration column chromatography. These results suggested that the native receptor protein was assemble by three 180 kDa protein. The N-terminal of 180 kDa protein was blocked by some residues, then the 180 kDa protein treated by a lysylendopeptidase and subsequently 6 oligopeptides were isolated from the digested solution by a HPLC.The N-terminal amino acid sequences of the oligopeptides were determined by a protein sequencer (Shimazu PSQ-1). The 180 kDa protein was a kind of glycosyl protein. Less

  • Research Products

    (5 results)

All Other

All Publications (5 results)

  • [Publications] T.Uemura,H.Ihara,A.Wadano,M.Himeno: "Fluorometric Assay of Potential Change of Bombyxmori Midgut Brush Border Membrane Induced by δ-Endotoxin from Ballus thuringiensis." Biosci.Biotech.Biochem.,. 56. 1976-1979 (1992)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] M.Himeno: "Mode of Action Bacillus thuringiensis Insectrcidal Toxin." International Symposium of University of Osaka Prefecture on Grobal Amenity,Proceedings. 425-432 (1992)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 姫野道夫: "感染症の生体防御の基本的原理:昆虫病原菌の生産する殺虫性タンパク質" 第67回日本細菌学会総会, 58-69 (1994)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Uemura, H.Ihara, A.Wadano, M.Himeno: "Fluoromtric Assay of Potential Change of Bombyx mori Midgut Brush Border Membrane Induced by delta-Endotoxin from Bacillus thuringiensis." Biosci.Biotech.Biochem.56 (12). 1976-1979 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Michio Himeno: "Mode of Action Bacillus thuringiensis Insecticidal Toxins." The Proceedings of The Inter.Symp.Univ.of Osaka Prefecture on Global Amenity.425-432 (1992)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1996-04-15  

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