Podocalyxinの構造と機能に関する薬理学的研究

• Project/Area Number: 01480136
• Research Category: Grant-in-Aid for General Scientific Research (B)
• Allocation Type: Single-year Grants
• Research Field: General pharmacology
• Research Institution: The University of Tokyo
• Principal Investigator: 遠藤 仁 東京大学, 医学部(医), 助教授 (20101115)
• Co-Investigator(Kenkyū-buntansha): 鄭 圭鎔 東京大学, 医学部・医学科, 外国人研究員
• Project Period (FY): 1989 – 1991
• Project Status: Completed (Fiscal Year 1991)
• Budget Amount: ¥6,800,000 (Direct Cost: ¥6,800,000); Fiscal Year 1991: ¥1,300,000 (Direct Cost: ¥1,300,000); Fiscal Year 1990: ¥2,200,000 (Direct Cost: ¥2,200,000); Fiscal Year 1989: ¥3,300,000 (Direct Cost: ¥3,300,000)
• Keywords: Podocalyxin / 限外濾過 / 陰性荷電障壁 / Puromycin aminonucleoside / Streptozotocin / Stainsーall染色 / 単離糸球体 / 単離系球体 / 糖タンパク / puromycin aminonucleosideネフロ-ゼ / stains-all染色

Research Abstract

平成2年度までに腎糸球体病態モデルにおけるpodocalyxinの病態生理学的意義を明らかにすることができたので、平成3年度にはpodocalyxinの構造解明と病態発症における役割の解析に向けて以下の実験を行った。
1)糸球体の単離と糖タンパクの可溶化。 ラット腎よりメッシュ法で糸球体を大量に単離し、0.2%Triton Xー100で可溶化した。この一部をSDS電気泳動とstains all染色でpodocalyxin存在を確認した。
2)WGA affinity column chromatographyによる部分精製。 前のステップ可溶化されたタンパクをWGAーsepharose 4Bと12時間4℃で反応させた後洗浄、Nーacetylーβーglucopyranosideで溶出させた。この分画をphosphotungstic acidで沈澱させ、acetoneで溶出を試みた。
3)ゲル濾過による精製 前のステップまでで数本の糖タンパクのバンドが得られており、podocalyxinも含まれていることは確認できたので、これ以降における単一バンドまでの精製を目下試している。最終的にはペプチドシ-ケンサ-分析に足る量の分取を目指している。以上の各段階での分取可能なタンパク質が限られているので、スタ-ティングマテリアルを増大させることに専念している。

Research Products

• [Publications] Endou,H.et al.: "Heterogeneity of nephron energy metabolism:Implications for response to hypoxic insult." Nephrology. 665-672 (1991)
• [Publications] Endou,H.et al.: "Role of ammonium ion in adenosine triphosphate consumption of the rat medullary thic ascending limb." Contr.Nephrol.92. 149-259 (1991)
• [Publications] Jung,K.Y.et al.: "A novel vasopressin receptor in rat early proximal tubule." Biochem.Biophys.Res.Commun.180. 131-137 (1991)
• [Publications] Endou,H.et al.: "Angiotensin II receptor subtypes in rat early proximal tubule." Contr.Nephrol.95. 190-196 (1991)
• [Publications] Endou,H.et al.: "Effect of mercuric chloride on angiotensin IIーinduced Ca^<++> transient in the proximal tubule of rats." Advances in Mercury Toxicology. 299-314 (1991)
• [Publications] Nosaka,K.et al.: "Cisplatinーinduced alterations in renal structure,ammoniagenesis and gluconeogenesis of rats." Kidney Int.41. 73-79 (1992)
• [Publications] Endou,H.et al.,eds.: "Molecular Aspects of Ammoniagenesis" Karger,Basel, 228 (1991)
• [Publications] Endou,H.et al.,eds.: "Cellular and Molecular Biology of the Kidney" Karger,Basel, 288 (1991)
• [Publications] Takemoto,F.et al.: "Intranephron PGE2 production in storokeーprone spontaneously hypertensive rats" Am.J.Physiol.258. H987-H993 (1990)
• [Publications] Endou,H.et al.: "Adenosime 2 receptor distribution along the rat nephron and its role in generation of reactive oxygen metabolites in glomeruli" The frontiers of nephrology. 163-171 (1990)
• [Publications] Jung,K.Y. et al.: "Cellular Adenosine Triphosphate Production and Consumption in the Descending Thin Limb of Henle's Loop in the Rat" Renal Physiol.Biochem.13. 248-258 (1990)
• [Publications] Jung,K.Y. et al.: "Furosemide Acts on Short Loop of Descending Thin Limb,But Not on Long Loop" J.Pharmacol.Exp.Ther.253. 1184-1188 (1990)
• [Publications] Satoh,T. et al.: "Inhibitory effect of phorbol ester on sodium transport in frog urinary bladder" Am.J. Physiol. 259. F425-F431 (1990)
• [Publications] Jung,K.Y. et al.: "Mercury chloride as a possible phospholipase C activator:Effect on angiotensin IIーinduced [Ca++]i transient in the rat early proximal tubule" Biochem.Biophys.Res.Commun.173. 606-613 (1990)
• [Publications] 遠藤 仁: "人体組織学呼吸器泌尿器(小川和朗、鈴木昭男、清寺真、永野俊雄、橋本一成、橋本正淑、山田英智、山元寅男、渡辺陽之輔編)" 朝倉書店, 415 (1990)
• [Publications] 遠藤 仁: "臨床腎臓病学(本田西男、小磯謙吉、黒川清編)" 朝倉書店, 584 (1990)
• [Publications] Endou,H.et al.: "Intranephron distribution of insulin binding in Wistar Kyoto and Spontaneously Hypertensive Rats" Proc.Japan Acad.(Ser.B). 65. 21-25 (1989)
• [Publications] Jung,K.Y.et al.: "Nephrotoxicity assessment by measuring cellulay ATP content I.Substrate specificities in the maintenance of ATP content in isolated rat nephron segments" Toxicol.Appl.Pharmacol.100. 369-382 (1989)
• [Publications] Jung,K.Y.et al.: "Nephrotoxicity assessment by measuring cellular ATP content II.Intranephron site of ochratoxin A nephrotoxicity" Toxicol.Appl.Pharmacol.100. 383-390 (1989)
• [Publications] Endou,H.et al.: "Modell zur Untersuchung nephrotoxischer Wirkungen" Arzeimitteltherapie. 7. 162 (1989)
• [Publications] Takahashi,T.et al.: "Intranephron distribution of purine-metabolizing enzymes in rats" Renal Physiol.Biochem.12. 287-294 (1989)
• [Publications] Miyanoshita,A.et al.: "Inhibitory effect of cyclic AMP on phorobol esterstimulated production of reactive oxygen metabolites in rat glomeruli" Biochem.Biophys.Res.Comm.165. 519-525 (1989)
• [Publications] Miyanoshita,A.et al.: "Furosemide directly stimulates prostaglandin E_2 prouction in the thick ascending limb of Henle's loop" J.Pharmacol.Exp.Ther.251. 1155-1159 (1989)
• [Publications] Jung,K.Y.et al.: "Biophasic increasing effect of angiotensin-II on intracellular free calcium in isolated rat early proximal tubule" Biochem.Biophys.Res.Comm.165. 1221-1228 (1989)
• [Publications] Endou,H.et al.: "Adenosine 2 receptor distribution along the rat nephron and its role in generation of oxygen metabolites in glomeruli" The Frontiers of nephrology. (1990)
• [Publications] Takemoto,F.et al.: "Intranephron PGE_2 production in stroke-prone spontaneously hypertensive rats" Am.J.Physiol.(1990)
• [Publications] Jung,K.Y.et al.: "Furosemide acts on short loop of descending thin limb,but not on long loop" J.Pharmacol.Exp.Ther.(1990)
• [Publications] Jung,K.Y.et al.: "Cellular ATP production and consumption in the descending thin limb of Henle's loop in the rat" Renal Physiol.Biochem.(1990)
• [Publications] 遠藤仁: "毒性生化学 上・下(佐藤哲男、上野芳夫、遠藤仁 編)" 地人書館, 623 (1989)
• [Publications] 遠藤仁: "臨床生理学シリ-ズ4 腎臓 (黒川清、今井正 編)" 南江堂, 315 (1989)