1992 Fiscal Year Annual Research Report

翻訳後修飾としてのチロシン-O-硫酸化とその機能(タンパク質チロシン硫酸転移酵素の遺伝子工学への応用)

Research Project

Project/Area Number	03660093
Research Institution	Miyazaki University
Principal Investigator	水光正仁宮崎大学, 農学部, 助教授 (00128357)
Keywords	硫酸化チロシン / 翻訳後修飾 / ゴルジ体 / 牛肝臓 / 硫酸転移酵素 / ヒルジン / 血液凝固阻害剤 / 活性硫酸
Research Abstract	生体内において硫酸化は、多くの外因性や内因性の化合物の代謝や、タンパク質のチロシン残基や糖鎖の翻訳後修師飾として重要な役割を演じている。これらの硫酸化は異なる硫酸転移酵素により活性硫酸3'-ホスホアデノシン5'-ホスホ硫酸(PAPS)から基質のOH基へ硫酸基を転移することにより行われる。種々の硫酸転移酵素は細胞質と膜の両面分に局在し、タンパク質チロシン硫酸化に関与する転移酵素(TPST)は、ゴルジ体の膜に結合する膜内在性糖タンパク質である。今回、牛肝臓ミクロゾーム画分およびゴルジ体に存在する硫酸転移酵素の諸性質の検討と遺伝子工学の産物すなわちリコンビナントタンパク質の効率良いチロシン硫酸化を目的として研究を行った。その結果、牛肝臓のゴルジ体から調製したTPSTはZ‐Glu‐TyrやBoc‐Cck‐8など数種のオリゴペプチドを硫酸化し、TyrのN端側がAspの時Gluに比べ効率よく硫酸化し、Lysの場合硫酸化しないことを明らかにした。このことよりTSPTの基質の認識はアミノ酸配列に依存していることが示唆された。またチロシンのC端のアミノ酸の疎水性の強さは、TPSTの基質認識にほとんど影響しなかった。また牛血清中の硫酸化チロシンに親和性をもつ蛋白質を反応混液に加えることにより、Km値が1/10に減少した。ヒル由来の血液凝固阻害剤ヒルジンは63番目のTyrのみ硫酸化を受けており、遺伝子工学的に酵母が生産したヒルジンは硫酸化されていないため、in vitroでの硫酸化が必要とされる。TPSTによる硫酸化を試みた結果、63番目のTyrのみの硫酸化に成功した。これらのことより、リコンビナントタンパク質の翻訳後修飾として、牛肝臓由来のTPSTを用いて特異的チロシン硫酸化が可能となった。

Research Products
(12 results)

All Other

All Publications (12 results)

[Publications] Ming-Chen Liu: "Possible Involvement of protein Tyrosine Sulrfation in the Polarized protein Targeting in Filter-Grown Madin-Darby Canine Kidney (MDCK) Cells." Animal Cell Technology. 1992. 463-471 (1992)
[Publications] P.H. Prasantha Fernando: "Protein Tyrosine Sulfation in Animal Cells and Its simulation in vitro in the Downstream Processing of Proteins." Animal Cell Technology. 1992. 473-479 (1992)
[Publications] Masahito Suiko: "Detoxication of Phenolic Xenobiotics in Microsomal Membranes: A Novel Phenol Sulfotransferase Activity in Bovine Liver Microsomes." The 6th China-Japan Symposium on Pesticide Science. 151-155 (1992)
[Publications] Hiroshi Ibuki: "Enzymatic Synthesis of PAPS with an ATP-regeneration System." Nucleic Acids Symposium Series. 27. 171-172 (1992)
[Publications] Masahito Suiko: "Post-translational Modification of Protein by Tyrosine Sulfation:Active Sulfate PAPS is the Essential Substrate for This Modification." Nucleic Acids Symposium Series. 27. 183-184 (1992)
[Publications] Masahito Suiko: "Quantitation of Tyrosine-O-sulfate in Human Urine by Ion-Pair Reverse-Phase High-Performance Liguid Chromatography." Bulletin of Faculty of Agriculture、Miyazaki University. 39. 141-146 (1992)
[Publications] P.H.Prasantha Fernando: "Protein Tyrosine Sulfation in Animal Cells and Its Simulation in vitro in the Downstream Processing of Proteins." Animal Cell Technology. 1992. 473-479 (1992)
[Publications] Masahito Suiko: "Detoxication of Phenolic Xenobiotics in Microsomal Membranes:A Novel Phenol Sulfotransferase Activity in Bovine Liver Microsomes." The 6th China-Japan Symposium Series. 151-155 (1992)
[Publications] Hiroshi Ibuki: "Enzymatic Synthesis of PAPS with an ATP-regeneration System." Nucleic Acids Symposium Series. 27. 171-172 (1992)
[Publications] Masahito Suiko: "Post-translational Modification of Protein by Tyrosine Sulfation:Active Sulfate PAPS is the Essential Substrate for This Modification." Nucleic Acids Symposium Series. 27. 183-184 (1992)
[Publications] Masahito Suiko: "Quantitation of Tyrosine-O-sulfate in Human Urine by Ion-Pair Reverse-Phase High-Performance Liguid Chromatography." Bulletin of Faculty of Agriculture、Miyazaki university. 39. 141-146 (1992)
[Publications] Masahito Suiko: "The Post-Trancelational Modification of Protein" Syozo Tuboi, Naoyuki Taniguchi and Nobuhiko Katumura, 281 (1992)

1992 Fiscal Year Annual Research Report

翻訳後修飾としてのチロシン-O-硫酸化とその機能(タンパク質チロシン硫酸転移酵素の遺伝子工学への応用)

Principal Investigator

水光 正仁 宮崎大学, 農学部, 助教授 (00128357)

Research Products

[Publications] Ming-Chen Liu: "Possible Involvement of protein Tyrosine Sulrfation in the Polarized protein Targeting in Filter-Grown Madin-Darby Canine Kidney (MDCK) Cells." Animal Cell Technology. 1992. 463-471 (1992)

[Publications] P.H. Prasantha Fernando: "Protein Tyrosine Sulfation in Animal Cells and Its simulation in vitro in the Downstream Processing of Proteins." Animal Cell Technology. 1992. 473-479 (1992)

[Publications] Masahito Suiko: "Detoxication of Phenolic Xenobiotics in Microsomal Membranes: A Novel Phenol Sulfotransferase Activity in Bovine Liver Microsomes." The 6th China-Japan Symposium on Pesticide Science. 151-155 (1992)

[Publications] Hiroshi Ibuki: "Enzymatic Synthesis of PAPS with an ATP-regeneration System." Nucleic Acids Symposium Series. 27. 171-172 (1992)

[Publications] Masahito Suiko: "Post-translational Modification of Protein by Tyrosine Sulfation:Active Sulfate PAPS is the Essential Substrate for This Modification." Nucleic Acids Symposium Series. 27. 183-184 (1992)

[Publications] Masahito Suiko: "Quantitation of Tyrosine-O-sulfate in Human Urine by Ion-Pair Reverse-Phase High-Performance Liguid Chromatography." Bulletin of Faculty of Agriculture、Miyazaki University. 39. 141-146 (1992)

[Publications] P.H.Prasantha Fernando: "Protein Tyrosine Sulfation in Animal Cells and Its Simulation in vitro in the Downstream Processing of Proteins." Animal Cell Technology. 1992. 473-479 (1992)

[Publications] Masahito Suiko: "Detoxication of Phenolic Xenobiotics in Microsomal Membranes:A Novel Phenol Sulfotransferase Activity in Bovine Liver Microsomes." The 6th China-Japan Symposium Series. 151-155 (1992)

[Publications] Hiroshi Ibuki: "Enzymatic Synthesis of PAPS with an ATP-regeneration System." Nucleic Acids Symposium Series. 27. 171-172 (1992)

[Publications] Masahito Suiko: "Post-translational Modification of Protein by Tyrosine Sulfation:Active Sulfate PAPS is the Essential Substrate for This Modification." Nucleic Acids Symposium Series. 27. 183-184 (1992)

[Publications] Masahito Suiko: "Quantitation of Tyrosine-O-sulfate in Human Urine by Ion-Pair Reverse-Phase High-Performance Liguid Chromatography." Bulletin of Faculty of Agriculture、Miyazaki university. 39. 141-146 (1992)

[Publications] Masahito Suiko: "The Post-Trancelational Modification of Protein" Syozo Tuboi, Naoyuki Taniguchi and Nobuhiko Katumura, 281 (1992)

水光正仁宮崎大学, 農学部, 助教授 (00128357)