Partial purification and reconstitution of inositol 1,4,5-trisphosphate receptor/Ca2+ channel of bovine liver microsomes

Hideaki Kamata, Masato Hirata, Shoichiro Ozaki, Iwao Kusaka, Yasuo Kagawa, Hajime Hirata

研究成果: ジャーナルへの寄稿学術誌査読

9 被引用数 (Scopus)

抄録

The binding of inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] to bovine liver microsomes was characterized. The Ins(1,4,5)P3 receptor of the microsomes was solubilized by 1% Triton X-100 and purified by sucrose density gradient, Heparin-Sepharose, DEAE-Toyopearl, ATP-Agarose, and Ins(1,4,5)P3-Sepharose column chromatographies. More than 1,000-fold enrichment of the Ins(1,4,5)P3-binding activity was achieved. Kd values of the binding activity were 2.8 nM in microsomes and 3.0 nM in the partially purified receptor, respectively, and the binding activity was optimal in the medium containing 100 mM KCl and at pH between 7.5 and 8.5. The presence of Ca2+ failed to inhibit the binding. Phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (Ptdlns), and phosphatidylinositol-4-monophosphate [PtdIns(4)P] showed no effect on the Ins(1,4,5)P3 binding. However, soybean phospholipids asolectin and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] strongly inhibited the binding activity. PtdIns(4,5)P2 inhibited the activity competitively with a half-maximal inhibitory concentration of 30 μg/ml. The partially purified Ins(1,4,5)P3 receptor was reconstituted into proteoliposomes. Fluorescence measurements using Quin 2 indicated that Ins(1,4,5)P3 stimulated Ca2+ influx into the proteoliposomes. The EC50 of Ins(1,4,5)P3 on Ca2+ influx was 50 nM. This result strongly suggest that Ins(1,4,5)P3 binding protein of liver microsomes acts as a physiological Ins(1,4,5)P3 receptor/Ca2+ channel.

本文言語英語
ページ(範囲)546-552
ページ数7
ジャーナルJournal of biochemistry
111
4
DOI
出版ステータス出版済み - 4月 1992

!!!All Science Journal Classification (ASJC) codes

  • 生化学
  • 分子生物学

フィンガープリント

「Partial purification and reconstitution of inositol 1,4,5-trisphosphate receptor/Ca2+ channel of bovine liver microsomes」の研究トピックを掘り下げます。これらがまとまってユニークなフィンガープリントを構成します。

引用スタイル