1997 Fiscal Year Annual Research Report

電気泳動ゲル内各種蛋白質燐酸化酵素の検出法の確立と応用

Research Project

Project/Area Number	08557012
Research Institution	Asahikawa Medical College
Principal Investigator	藤澤仁旭川医科大学, 医学部, 教授 (10027039)
Co-Investigator(Kenkyū-buntansha)	石田敦彦旭川医科大学, 医学部, 助手 (90212886) 亀下勇旭川医科大学, 医学部, 助教授 (60127941)
Keywords	ゲル電気泳動法 / アッセイ方法 / 蛋白質燐酸化酵素 / 蛋白質脱燐酸化酵素 / 合成基質ペプチド / シグナル伝達 / 蛋白質燐酸化反応 / 蛋白質脱燐酸化反応
Research Abstract	1.合成オリゴペプチドを基質に用いた電気泳動ゲル内蛋白質燐酸化酵素の検出法の開発と応用我々が1989年に報告した電気泳動ゲル内蛋白質燐酸化酵素検出法に合成オリゴペプチドを蛋白質燐酸化酵素の基質として用いる方法を検討し、本検出法の汎用化を目指した。合成オリゴペプチドは単独ではゲル内に固定できないが、ポリリジンに共有結合させると効率よく固定され、蛋白質燐酸化酵素の基質となる特異配列が認識されることが明らかになった。この方法を用いてMAPキナーゼによって燐酸化されるミエリン塩基蛋白質の燐酸化部位周辺のアミノ酸配列を持つ合成ペプチドをゲル内に固定し、細胞の粗抽出液中のMAPキナーゼ活性を特異的にかつ、高感度で検出できることを確認した。 2.電気泳動ゲル内蛋白質脱燐酸化酵素の検出法の開発と応用ポリリジンに結合させた合成オリゴペプチドを蛋白質燐酸化酵素で燐酸化した後、ゲル内に固定して蛋白質脱燐酸化酵素を検出する方法を開発した。この方法を用いてCaMキナーゼIIの自己燐酸化部位周辺のアミノ酸配列を持つ合成ペプチドをCaMキナーゼIIで燐酸化した後ゲル内に固定し、ラットの脳の粗抽出液中の脱燐酸化酵素をスクリーニングした。CaMキナーゼIIの自己燐酸化部位を特異的に脱燐酸化する新しい蛋白質脱燐酸化酵素が検出されたので、この脱燐酸化活性を追跡しながらラットの脳からCaMキナーゼII脱燐酸化酵素を純粋に精製し、その性質を明らかにした。

Research Products

(14 results)

All Other

All Publications (14 results)

[Publications] S.Okuno: "Purification and Characterization of Ca^<2+>/Calmodulin-Dependent Protein Kinase Kinase β from Rat Cerebellum" J.Biochem.121(1). 155-160 (1997)
[Publications] I.Kameshita: "Detection of Protein Phosphatase Activities in Sodium Dodecyl Sulfate-Polyacrylamide Gel Using Peptide Substrates" Anal.Biochem.245. 149-153 (1997)
[Publications] T.Kitani: "Studies on the Site of Phosphorylation of Ca^<2+>/Calmodulin-Dependent Protein Kinase(CaM-Kinase)IV by CaM-Kinase Kinase" J.Biochem.121. 804-810 (1997)
[Publications] T.Kiatani: "Molecular Cloning of Ca^<2+>/Calmodulin-Dependent Protein Kinase Kinase β" J.Biochem.122(1). 243-250 (1997)
[Publications] S.Okuno: "Studies on the Substrate Specificity of Ca^<2+>/Calmodulin-Dependent Protein Kinase Kinase α" J.Biochem.122. 337-343 (1997)
[Publications] I.Kameshita: "Detection of Calcium Binding Proteins by Two-Dimensional Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis" Anal.Biochem.249. 252-255 (1997)
[Publications] I.Kameshita: "A New Peptide Conjugate as a Highly Specific Substrate for MAP Kinase" J.Biochem.122(1). 168-172 (1997)
[Publications] M.Takeuchi: "Perinuclear Membrane Localization of α KAP,a Protein Produced from a Gene within the Gene of Calmodulin-Dependent Protein Kinase II α" J.Biochem.122(3). 494-497 (1997)
[Publications] Y.Hayashi: "Calcium-and calmodulin-dependent phosphorylation of AMPA type glutamate receptor subunits by endogenous protein kinases in the post-synaptic density" Mol.Brain Res.46. 338-342 (1997)
[Publications] S.Okuno: "Regulation of Ca^<2+>/Calmodulin-Dependent Protein Kinase IV(CaM-Kinase IV)by Changing Its Susceptibility to Phosphorylation by CaM-Kinase Kinases" J.Biochem.122(5). 897-900 (1997)
[Publications] A.Ishida: "Assay of Protein Phosphatase Activities with Phosphopeptide-Magnetic Particle Conjugates" Anal.Biochem.254. 152-155 (1997)
[Publications] A.Ishida: "A Novel Protein Phosphatase That Dephosphorylates and Regulates Ca^<2+>/Calmodulin-dependent Protein Kinase II" J.Biol.Chem.273(4). 1904-1910 (1998)
[Publications] 奥野幸子: "蛋白質・核酸・酵素" 共立出版, 6 (1997)
[Publications] A.Ishida: "Kinases and Phosphatases in Lymphocyte and Neuronal Signaling" Spring-Verlag Tokyo, 2 (1997)

1997 Fiscal Year Annual Research Report

電気泳動ゲル内各種蛋白質燐酸化酵素の検出法の確立と応用

Principal Investigator

藤澤 仁 旭川医科大学, 医学部, 教授 (10027039)

Research Products

[Publications] S.Okuno: "Purification and Characterization of Ca^<2+>/Calmodulin-Dependent Protein Kinase Kinase β from Rat Cerebellum" J.Biochem.121(1). 155-160 (1997)

[Publications] I.Kameshita: "Detection of Protein Phosphatase Activities in Sodium Dodecyl Sulfate-Polyacrylamide Gel Using Peptide Substrates" Anal.Biochem.245. 149-153 (1997)

[Publications] T.Kitani: "Studies on the Site of Phosphorylation of Ca^<2+>/Calmodulin-Dependent Protein Kinase(CaM-Kinase)IV by CaM-Kinase Kinase" J.Biochem.121. 804-810 (1997)

[Publications] T.Kiatani: "Molecular Cloning of Ca^<2+>/Calmodulin-Dependent Protein Kinase Kinase β" J.Biochem.122(1). 243-250 (1997)

[Publications] S.Okuno: "Studies on the Substrate Specificity of Ca^<2+>/Calmodulin-Dependent Protein Kinase Kinase α" J.Biochem.122. 337-343 (1997)

[Publications] I.Kameshita: "Detection of Calcium Binding Proteins by Two-Dimensional Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis" Anal.Biochem.249. 252-255 (1997)

[Publications] I.Kameshita: "A New Peptide Conjugate as a Highly Specific Substrate for MAP Kinase" J.Biochem.122(1). 168-172 (1997)

[Publications] M.Takeuchi: "Perinuclear Membrane Localization of α KAP,a Protein Produced from a Gene within the Gene of Calmodulin-Dependent Protein Kinase II α" J.Biochem.122(3). 494-497 (1997)

[Publications] Y.Hayashi: "Calcium-and calmodulin-dependent phosphorylation of AMPA type glutamate receptor subunits by endogenous protein kinases in the post-synaptic density" Mol.Brain Res.46. 338-342 (1997)

[Publications] S.Okuno: "Regulation of Ca^<2+>/Calmodulin-Dependent Protein Kinase IV(CaM-Kinase IV)by Changing Its Susceptibility to Phosphorylation by CaM-Kinase Kinases" J.Biochem.122(5). 897-900 (1997)

[Publications] A.Ishida: "Assay of Protein Phosphatase Activities with Phosphopeptide-Magnetic Particle Conjugates" Anal.Biochem.254. 152-155 (1997)

[Publications] A.Ishida: "A Novel Protein Phosphatase That Dephosphorylates and Regulates Ca^<2+>/Calmodulin-dependent Protein Kinase II" J.Biol.Chem.273(4). 1904-1910 (1998)

[Publications] 奥野幸子: "蛋白質・核酸・酵素" 共立出版, 6 (1997)

[Publications] A.Ishida: "Kinases and Phosphatases in Lymphocyte and Neuronal Signaling" Spring-Verlag Tokyo, 2 (1997)

藤澤仁旭川医科大学, 医学部, 教授 (10027039)