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Mass spectrometry of phosphatidylinositols with various fatty acid side chain

Research Project

Project/Area Number 09672138
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Chemical pharmacy
Research InstitutionThe Universityu of Tokyo

Principal Investigator

MORISAKI Naoko  Institute of Molecular and Cellular Biosciences, The University of Tokyo, Lecturer, 分子細胞生物学研究所, 講師 (00092354)

Co-Investigator(Kenkyū-buntansha) SHIRAI Ryuichi  Institute of Molecular and Cellular Biosciences, The University of Tokyo, Resear, 分子細胞生物学研究所, 助手 (80183838)
Project Period (FY) 1997 – 1998
Project Status Completed (Fiscal Year 1998)
Budget Amount *help
¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1998: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1997: ¥1,200,000 (Direct Cost: ¥1,200,000)
Keywordsphosphatidylinositol / phosphatidylinositol 3-kinase / matrix / FAB MS / phosphatidylinositol 3-phosphate / ホスファチジルイノシトール 3-キナーゼ / 負イオンFABMS / ホスファチジルイノシトール 3-リン酸 / 情報伝達 / 脂肪酸側鎖 / PI3キナーゼ / PI3 5-ホスファターゼ / 質量分析
Research Abstract

Phosphatidylinositol 3-kinase (Pl 3-kinase) is a key enzyme in the signaling pathways of 3-phosphorylated polyphosphoinositides. The major substrate of Pl 3-kinase, in vivo, is assumed to be phosphatidylinositol 4, 5-bisphosphate (Pl 4, 5-P_2), generating Pl 3, 4, 5-P_3, a putative second messenger to play important roles in signal transduction. In vitro, the enzyme phosphorylates Pl, Pl 4-P and Pl 4, 5-P_2, The natural Pl purified from animal tissues, which is an excellent substrate of Pl 3-kinase, contains arachidonate at the sn-2 position. On the other hand, we found that synthetic Pls with saturated fatty acid of carbon number 18 and 20 at the sn-2 position, were not phosphorylated by the enzyme. In order to evaluate the phosphorylation reaction with Pl 3-kinase, we optimized the sn-2 fatty acids of Pl.
A mixture of Pls (Pl_<C4>, Pl_<C6>, Pl_<C8>, Pl_<C10>, Pl_<C12>, Pl_<C14>, Pl_<C16>) with various saturated fatty acids at the sn-2 center was synthesized from an equimolar mixture of seven carboxylic acids (C4, C6, C8, C10, C12, C14, C16), and was subjected to the enzymatic reaction with Pl 3-kinase. The product was analyzed by negative ion fast atom bombardment (FAB) mass spectrometry using a matrix (triethanolamine : glycerol = 3 : 1). The spectrum showed the [M-H] ion peaks of monophosphorylated Pl_<C4>, Pl_<C6>, Pl_<C8> and Pl_<C10> analogs. The phosphorylated ions derived from the longer chain analogs such as Pl_<C16> were detected only at trace levels. Short sn-2 fatty acid analogs such as Pl_<C4>, Pl_<C8> were also shown to be excellent substrates of Pl 3-kinase by independent phosphorylation of synthetic Pls (Pl_<C2>, Pl_<C4>, Pl_<C8>, Pl_<C12>, Pl_<C16>, Pl_<C18>) with Rl 3-kinase and [gamma-^<32>P]-ATP.
These results are applicable for the design of artificial second messenger molecules, enzyme inhibitors, affinity probes to find specific binding proteins and photoaffinity labeling probes.

Report

(3 results)
  • 1998 Annual Research Report   Final Research Report Summary
  • 1997 Annual Research Report
  • Research Products

    (14 results)

All Other

All Publications (14 results)

  • [Publications] Sawada,T. 他: "Efficient a symmetric synthesis of phosphatidyl-D-myo-inositol 3.4.5-triphosphate" Chem.Pharm.Bull.45. 1521-1523 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Tanaka,K. 他: "A target of phosphatidylinositol 3.4.5-trisphosphate with a zink finger motif similar to that of the ADP-vibosylation-factor..." Eur.J.Biochem.245. 512-519 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Shirai,T. 他: "specific detection of phosphatidylinositol 3.4.5-trisphosphate binding proteins by the PlP3 analogue beads : an application..." Biochim.Biophys.Acta. 1402. 292-302 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Shirai.R.,他: "Synthesis of diacye glycerol analogs of phosphatidylinositol 3.4.5-tris phosphate" Tetahadron Lett.39. 9485-9488 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Shirai,R.他: "The Structural requirement of phosphatidylinositols as substrate of phosphatidylinositol 3-kinase" Tetrahedion Lett.40. 1693-1696 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] K.Tanaka, et al.: "A target of phosphatidylinositol 3,4,5-trisphosphate with a zinc finger motif similar to that of the ADP ribosylation factor GTPase activating protein and two pleckstrin homology domains" Eur.J.Biochem.245. 512-519 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] T.Sawada, et al.: "Efficient asymmetric synthesis of phosphatidyl-D-myo-inositol3,4,5-trisphosphate" Chem.Pharm.Bull.45. 1521-1523 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] T.Ken-ichi, et al.: "Specific detection of phosphatidylinositol3,4,5-trisphosphate binding proteins by the PIP3 analogue beads : An application for rapid purification of the PIP3 binding proteins" Biochim.Biophys.Acta. 1402. 292-302 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] R.Shirai, et al.: "Synthesis of diacylglycerol analogs of phosphatidylinositol3,4,5-trisphosphate" Tetrahedron Lett. 39. 9485-9488 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] R.Shirai, et al.: "The structural requirement of phosphatidylinositols as substrate of phosphatidylinositol3-kinase" Tetrahedron Lett. 40. 1693-1696 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1998 Final Research Report Summary
  • [Publications] Shirai,T.他: "Specific deteation of phosphatidylinositol 3.4.5-trisphosphats binding proteins by the P1P_3 analogue beads : an application ・・・" Biochim.Biophys.Acta. 1402. 292-302 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Shirai,R.他: "Synthesis of diacyeglycerol analogs of phosplratidylinositol 3・4・5・trisphosphate" Tetrahedron hett.39. 9485-9488 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Shirai,R.他: "The structural requirement of phosphatidyeinositols as substrate of phosphatidylinositol 3・kinase" Tetrahedron hett.40. 1693-1696 (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] K.Tanakaら: "A Target of Phosphatidyl 3,4,5-Triphosphate with 〜" Eur.J.Biochem.245.512-519 (1997)

    • Related Report
      1997 Annual Research Report

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Published: 1997-04-01   Modified: 2016-04-21  

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