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New Type of Particulate Guanylate Cyclase, ATP/DTT-Stimulated Guanylate Cyclase : Some Proparties and the Regulation of its Activity, and Pathophysiological Roles

Research Project

Project/Area Number 11670091
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field General pharmacology
Research InstitutionSaga Medical School

Principal Investigator

ASAKAWA Takeo  Saga Medical School, Dept. of Pharmacology, Professor, 医学部, 教授 (50028362)

Co-Investigator(Kenkyū-buntansha) OKAMOTO Hideaki  Saga Medical School, Dept. of Pharmacology, Research Assistant, 医学部, 助手 (70315183)
TAKANO Masako  Saga Medical School, Dept. of Pharmacology, Research Assistant, 医学部, 助手 (00154807)
Project Period (FY) 1999 – 2001
Project Status Completed (Fiscal Year 2001)
Budget Amount *help
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2001: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2000: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1999: ¥1,400,000 (Direct Cost: ¥1,400,000)
Keywordsquanylate cyclase / cyclic GMP / ATP / signal transduction / cyclic nucleotide / 環状ヌクリオチド
Research Abstract

1) Novel ATP/DTT-Stimulated GC; ATP/DTT-stimulated guanylate cyclass (GC) in lung membrane was stimulated 18-fold by ATP and DTT, and both its activity and ANP-stimulated GC activity were observed to be additive. ATP/DTT-stimulated GC was solubilized by octyl glucoside (OG) to examine the mechanism of ATP/DTT-stimulation. GC in OG-extracts was stimulated maximally 2.5-fold by both ATP, ATPγS or AMPPNP, and DTT. Preincubation of OG-extracts at 10ーC with AMPPNP and DTT (Ist-preincubation) converted GC to an insensitive state to stimulation by both ATP and DTT, and this conversion was partly inhibited by a proteinphosphatase-1 inhibitor (10-1000 nM okadaic acid). On the other hand, ANP-stimulated GC was not converted to an insensitive state to ANP/ATP-stimulation by the 1st-preincubation. And, subsequent preincubation of OG-extracts at 10℃ with both DTT and, ATP or ATPγS but not AMPPNP converted GC to a state sensitive to ATP/DTT-stimulation, and this conversion was partly inhibited by in … More hibitors of Ca^<2+>/calmodulin-dependent protein kinase II (KN 62 and KN-93). In contrast, the preincubation with KN-62 and KN-93 had no effect on ANP-stimulated GC activity. The results suggested that phosphorylation was involved in the regulation of ATP/DTT-stimulated GC sensitivity to ATP/DTT-stimulation, and that ATP/DTT-stimulated GC activity was likely to be a different type from ANP-stimulated GC activity.
2) Measurement of Membrane Protein; Accurate determination of low levels of protein in samples containing large amounts of interfering substances is rather difficult. In this report, we described an improved method, the DOC-TCA-washing-BCA method, which enabled us to perform rapid and efficient removal of many interfering substances, and to allow proteins to be detected above 0.05μg.
3) New variant of β2 Subunit of Soluble GC (β2b) ; We isolated two distinct cDNA fragments for the β2 subunit of soluble GC (β2a and β2b) from a rat brain cDNA library. The deduced amino acid sequence of β2b is C-terminally shorter by 46 amino acids and thus does not contain a consensus sequence for isoprenylation / carboxymethylation. RT-PCR analysis demonstrated that both variants are expressed in various tissues, including kidney, liver, and brain. Less

Report

(4 results)
  • 2001 Annual Research Report   Final Research Report Summary
  • 2000 Annual Research Report
  • 1999 Annual Research Report
  • Research Products

    (32 results)

All Other

All Publications (32 results)

  • [Publications] Shiwen Luo: "Regulation of Guanylate Cyclase by ATP and Dithiothreitol in Rat Lung Membrane : Involvement of an Insensitive and a Sensitive State to ATP/Dithiothreitol-Stimulation"The Japanese Journal of Pharmacology. (In press). (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Takeo Asakawa: "Possible involvement of protein phosphorylation and dephosphorylation in the regulation of DTT-and ATP-dependent guanylate cyclase"The Japanese Journal of Pharmacology. 88・Sup.I. 167 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Shiwen Luo: "新規膜結合型グアニル酸シクラーゼ,SAT-GCの活性調節の分子機構"日本薬理学雑誌. 119・3. 61 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Shiwen Luo: "Bicin choninic acid (BCA)法による可溶化膜タンパク質の微量定量-妨害物質の影響を除去する方法の考案"生化学. 73・8. 795 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Takeo Asakawa: "Measurement of solubilized membrane-protein, using bicinconinic acid (BCA):a device for elimination of interfering substances"The Japanese Journal of Pharmacology. 85・Sup.I. 185 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Shiwen Luo: "Purification and some properties of the activation of membrane-bound guanylate cyclase, SAT-GC of rat lung"The Japanese Journal of Pharmacology. 85・Sup.I. 181 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Hideaki Okamoto: "Molecular cloning and expression analysis of a novel splice variant of rat sGC β2 subuit"The Japanese Journal of Pharmacology. 85・Sup.I. 196 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Shiwen Luo: "新しいタイプの膜結合型グアニル酸シクラーゼ,SAT-GCの可溶化部分精製と,それに伴う膜タンパク質の微量定量法"日本薬理学雑誌. 117・3. 75 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] 高野正子: "CFG-GC(膜結合型グアニル酸シクラーゼ)の活性化におけるGTP要求性について"生化学. 72・8. 992 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Shiwen Luo: "Purification and the effect of sphingolipids on the activity of particulate, SAT-guanylate cyclase of rat lung"The Japanese Journal of Pharmacology. 82・Sup.I. 145 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] 頼 仲平: "新しいタイプの膜結合型グアニル酸シクラーゼ活性,SAT-GCへのSphingolipidの効果について"日本薬理学雑誌. 115・3. 80 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Takeo Asakawa: "Properties of GCM and the effect of sphingolipid on membrane-bound guanylate cyclase of rat brain and lung"The Japanese Journal of Pharmacology. 79・Sup.I. 68 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] 高野正子: "新しいタイプの膜結合型グアニル酸シクラーゼ活性へのSphingolipidの効果について"生化学. 71・8. 899 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] S. Luo, M. Takano and T. Asakawa: "Regulation of guanylate Cyclase by ATP and Dithiothereitol in Rat Lung Membrane : Involvement of an Insensitive and a Sensitive State to ATP/Dithiothreitol-Stimulation"The Japanese Journal of Pharmacology. (in press). (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] T. Asakawa, S. Luo and M. Takano: "Possible involvement of protein phosphorylation and dephosphorylation in the regulation of DTT-and ATP-dependent quanylate cyclase"The Japanese Journal of Pharmacology. 88・Sup.I. 167 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] S. Luo, M. Takano and T. Asakawa: "Measurement of solubilized membrane-protein, using bicinconinic acid (BCA) : a device for elimination of interfering substances"The Japanese Journal of Pharmacology. 85・Sup.I. 185 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] S. Luo, M. Takano and T. Asakawa: "Purification and some properties of the activation of membrane-bound quanylate cyclase, SAT-GC of rat lung"The Japanese Journal of Pharmacology. 85・Sup.I. 181 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] H. Okamoto and T. Asakawa: "Molecular cloning and expression analysis of a novel splice variant of rat sGC β2 subuit."The Japanese Journal of Pharmacology. 85・Sup.I. 196 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] S. Luo, M. Takano, Z. Lai and T. Asakawa: "Purification and the effect of sphingolipids on the activity of particulate, SAT-qanylate cyclase of rat lung"The Japanese Journal of Pharmacology. 82・Sup.I. 145 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Shiwen Luo: "Regulation of Guanylate Cyclase by ATP and Dithiothreitol in Rat Lung Membrane : Involvement of an Insensitive and a Sensitive State to ATP/ Dithiothreitol-Stimulation"The Japanese Journal of Pharmacology. (In press). (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] Takeo Asakawa: "Possible involvement of protein phosphorylation and dephosphorylation in the regulation of DTT-and ATP-dependent guanylate cyclase"The Japanese Journal of Pharmacology. 88・Sup.I. 167 (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] Shiwen Luo: "新規膜結合型グアニル酸シクラーゼ,SAT-GCの活性調節の分子機構"日本薬理学雑誌. 119・3. 61 (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] Shiwen Luo: "Bicinchoninic acid(BCA)法による可溶化膜タンパク質の微量定量-妨害物質の影響を除去する方法の考案"生化学. 73・8. 795 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Takeo Asakawa: "Measurement of solubilized membrane-protein, using bicinconinic acid (BCA) : a device for elimination of interfering substances."The Japanese Journal of Pharmacology. 85・SupI. 185-185 (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Shiwen Luo: "Purification and some properties of the activation of membrane-bound guanylate cyclase, SAT-GC of rat lung."The Japanese Journal of Pharmacology. 85・SuppI. 181-181 (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Hideaki Okamoto: "Molecular cloning and expression analysis of a novel splice variant of rat sGC β2 subuit."The Japanese Journal of Pharmacology. 85・SupI. 196-196 (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Shiwen Luo: "新しいタイプの膜結合型グアニル酸シクラーゼ,SAT-GCの可溶化部分精製と,それに伴う膜タンパク質の微量定量法"日本薬理学雑誌. 117・3. 75-75 (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] 高野正子: "CFG-GC(膜結合型グアニル酸シクラーゼ)の活性化におけるGTP要求性について"生化学. 72・8. 992-992 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Takeo Asakawa: "Properties of GCM and the effect of sphingolipid on membrane-bound guanylate cyclase of rat brain and lung"The Japanese Journal of Pharmacology. 79・SupI. 68 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Shiwen Luo: "Purification and the effect of sphingolipids on the activity of particulate,SAT-guanylate cyclase of rat lung"The Japanese Journal of Pharmacology. 82・SupI. 145 (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] 高野正子: "新しいタイプの膜結合型グアニル酸シクラーゼ活性へのSphingolipidの効果について"生化学. 71・8. 899 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] 頼 仲平: "新しいタイプの膜結合型グアニル酸シクラーゼ活性,SAT-GCへのSphingolipidの効果について"日本薬理学雑誌. 115・3. 80 (2000)

    • Related Report
      1999 Annual Research Report

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Published: 1999-04-01   Modified: 2016-04-21  

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