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Molecular structural analysis of membrane proteins incorporated into liposomes or expressed transitionally in the endoplasmic reticulum of transfected COS cells

Research Project

Project/Area Number 06670041
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field General anatomy (including Histology/Embryology)
Research InstitutionKansai Medical University

Principal Investigator

WATANABE Jun  Kansai Medical University, School of Medicine, Associate Professor, 医学部, 助教授 (40148557)

Project Period (FY) 1994 – 1996
Project Status Completed (Fiscal Year 1996)
Budget Amount *help
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1996: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1995: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1994: ¥1,000,000 (Direct Cost: ¥1,000,000)
KeywordsEndoplasmic reticulum / Liposome / Cytochrome P-450 / Atomic force microscope / Hepatocytes / 肝細胞 / 生体膜 / リポソーム / チトクローム P-450 / チトクローム p-450環元酵素 / 膜蛋白 / チトクローム-P450還元酵素
Research Abstract

To analyze the structure of membrane-bound proteins in the endoplasmic reticulum (ER) of rat hepatocytes at subnanomicrometer level, cytochrome P-450 (P-450) IIB and P-450 reductase incorporated into liposomes or expressed transitionally in the ER of COS cells were examined by atomic force microscopy and freeze-fracture replica method followed by electron microscopy. Expression of P-450 IIB in COS cells transfected P-450 IIB2 gene was various among experiments, and the number of incorporated molecules in ER was uncontrollable. Thus, this system was inadequate for the present purpose. Alternatively, liposomes made by the detergent eliminating method (Mimms et al., 1981), the freeze-thawing method (Pick, 1981) and the freeze-thawing-dialysis method (Oku & MacDonald, 1983) gave good results ; large unilamellar liposomes that mimic the membrane of the endoplasmic reticulum were obtained. Liposomes produced by the three methods are thus suitable for the analysis of the interaction between the endoplasmic reticulum membrane and membrane-bound proteins. The number of incorporated proteins could be controlled by fusing proteoliposomes with liposomes. Then, to examine the relationship between the density of cytochrome P-450 (P-450) molecules in the ER and distribution of the molecules in ER,P-450 IIB2 molecules purified from rat livers were incorporated into liposomes made from the mixture of phospholipids in which the phospholipid composition was similar to that in ER by the detergent elimination followed by freeze-thawing. The incorporated liposomes were exploded with distillled water and the resulting planar membrane fragments were examined. When large amount of P-450 molecules were incorporated, many particles were scattered in the membrane fragments. If the incorporated P-450 molecules was decreased, the particles decreased and formed clusters.

Report

(4 results)
  • 1996 Annual Research Report   Final Research Report Summary
  • 1995 Annual Research Report
  • 1994 Annual Research Report
  • Research Products

    (32 results)

All Other

All Publications (32 results)

  • [Publications] Watanabe,Jun: "Measurement of NAPPH-cytochrome P-450 reductase content in rutliver sections by quantitutive immunohisro chemistry with a vicleo imaye prosessor." J.Histochem.Cytochem.42. 1161-1167 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Watanabe,Jun: "The absence of paravascular merve projection and cross innerration in interscapulur brown achpose tissues of mice." J.Auton.Nerve.Syst.49. 269-276 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Amatsu,Tsuyoshi: "Endoplasmic reticulum proliferates without an incrense in cytochrome P-450 in bepatocytes of mice treuted with phemburbital and cobalt chleride." Eur.J.Cell Biol.68. 256-262 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Watanabe,Jun: "Relutionship between immunostaining intensty and antiycn cuntent insections" J.Histochem.Cytochem.44. 1451-1458 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Watanabe,Jun: "Preparation of liposumes that mimic the membrune of encluplrsmic retiwlum of rat hepato cytes" J.Electron Microsc.45. 171-176 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Watanabe,Jun: "Peri-and postnatal changes in reduced nicotinamide adenine dinucleotide phosphate-cytochrume P-450 rontent in heputaytes of rats." Histochem.J.28. 505-510 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Watanabe J,Asaka Y,Tanaka T and Kanamura S: "Measurement of NADPH-cytochrome P-450 reductase content in rat liver sections by quantitative immunohistochemistry with a video image processor" J.Histochem.Cytochem.42. 1161-1167 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Watanabe J,Mishiro K,Amatsu T and Kanamura S: "The absence of paravascular nerve projection and cross innervation in interscapular brown adipose tissues of mice" J.Auton.Nerve.Syst.49. 269-276 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Amatsu T,Watanabe J,Asaka Y and Kanamura S: "Endoplasmic reticulum proliferates without an increase in cytochrome P-450 in hepatocytes of mice treated with phenobarbital and cobalt chloride" Eur.J.Cell Biol.68. 256-262 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Watanabe J: "Cytochrome P-450 reductase content in rat liver sections by quantitative immunohistochemistry with video image processor" J.Histochem.Cytochem.43. 561 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Watanabe J,Asaka Y,Mino K and Kanamura S: "Selective transport of glycolipid molecules containing N-acetylgalactosamine to the basolateral domain of mouse hepatocytes plasma membrane" Acta Histochem.Cytochem.28. 357-364 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Takana J-I,Watanabe J,Mishiro K,Kanamura S: "Visualization of acetylcholine in the mouse brain by a combination of immunohistochemistry with ionic fixation" Acta Histochem.Cytochem.28. 231-237 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Watanabe J,Asaka Y,Kanamura S: "Relationship between immunostaining intensity and antigen content in sections" J.Histochem.Cytochem.44. 1451-1458 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Watanabe J,Asaka Y,Mino K and Kanamura S: "Preparation of liposomes that mimic the membrane of endoplasmic reticulum of rat hepatocytes" J.Electron Microsc.45. 171-176 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Watanabe J,Asaka Y and Kanamura S: "Peri- and postnatal changes in reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase content in hepatocytes of rats" Histochem.J.28. 505-510 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Watanabe,Jun: "Relationship between immunostaining imtensity and antigen content in sections." J、Histochem. Cytochem.44. 1451-1458 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Watanabe,Jun: "Preparation of liposumes that mimic the membrane of endoplamic reticulum of rat hepatocytes." J. Electron Microsc.45. 171-176 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Watanabe,Jun: "Peri-and postnatal changes in reduced nicotinamide adenine clinucleotide phosphate-cytochrome P-450 reductase cuntont in hepatocytes of rats." Histochem. J.28. 505-510 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Watanabe.J.: "Relationship between lmmtostaining intensity of peroxiduc-antiperoeldnse method or that of avidin-biotin cunples methoel insections and concentretion of prinary untibody" Acts Histochem. Cytochem.29. S401-S401 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Asaka,Youko: "Localization of xenobiotic responsire element binding factor in rot hepatocytes as revealed by in situ Sowthuestorn hybridization." Acts Histochem. Cytochem.29. S824-S825 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] 金村泰輔: "組織細胞化学1996" 学際企画, 208 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Watanabe, Jun et al.: "Selective transport of glycolipid molecules containing N-acetylgalactosamine to the basuluteral clomain of mouse hepatocyte plusma menbrane" Acta Histochem. Cytochem.28. 357-364 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Watanabe, Jun: "Cytochrome P-450 reductase content in rat liver sections by guantitative immunobisto of emistry with video image processur : avtbys' response" J. Histochem, Cytochem. 43. 561 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Amatsu, Tsuyoshi et al: "Endoplasmic reticulum prolifevates without an increase in cytochrume P-450 in heputocytes of mice treated with phenobarbital and cobult chloricle" Eur. J. Cell Biol.68. 256-262 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Tanaka, Jun-ichi et al.: "Visualization of acetylchollne in the mouse brain by a combination of immnobiotochemistry with ionic fixatin." Acta Histochem. Cytochem. 28. 231-237 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Watanabe, Jun etal: "Preparation of liposunes that mimic the membrune of endoplasmic reticulum of rat bepatocyte" J. Electron Microsc. 45(印刷中). (1996)

    • Related Report
      1995 Annual Research Report
  • [Publications] Watanabe,Jun: "Measurement of NADPH-cytochrome P-450 reductase content in rat liver sections by quantitative immunohistochemistry with a video image processr." J.Histochem.Cytochem.42. 1161-1167 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Watanabe,Jun: "Absence of paravascular nerve projection and cross-innervation in interscapular brown aclipose tissues of mice" J.Auton.Nerv.Syst.49. 269-276 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Fujimoto,Satoshi: "Age-related changes in fiber number,fiber size,fiber type composition and adenosine triphosphatase activity in rat soleus muscle" Ann.Anat.176. 429-435 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] 渡辺 淳: "光学顕微鏡定量分析と電子顕微鏡形態計測法" 電子顕微鏡. 29. 54-57 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] 渡辺 淳: "形態計測と顕微分光測光による細胞内分子密度の測定" 細胞. 26. 273-276 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Watanabe,Jun: "Cytochrome P-450 reductase content in rat liver sections by quantitutive immunohistochemistry with vicleo image processor:Authors′response." J.Histochem.Cytochem.43(印刷中). (1995)

    • Related Report
      1994 Annual Research Report

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Published: 1994-04-01   Modified: 2016-04-21  

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