Intracellular Calcium Ion Signal Transduction System in Plant

植物细胞内钙离子信号转导系统

• 批准号: 01480010
• 负责人: TORIYAMA Shoshi
• 金额: $ 4.42万
• 依托单位: University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-01480010/
• 关键词: Calcium ion; Phosphoinositides; Phospholipase C; Inositol trisphosphate; Ca^<2+>-pump; Ca^<2+>-channel; Ca^<2+>-dependent protein kinase; Patch-clamp method; フオスフォリパ-ゼC; GTP結合蛋白; 電圧依存性イオンチャネル; Ca^<2+>ーチャネル; Ca^<2+>ーポンプ; イノシト-ルミリン酸; フォスファチジルイ

(1) Enzymes involved in metabolic turnover of inositolphospholipid, phosphatidyl inositol kinase, phosphatidylinositol-monophosphate kinase and phospholipase C (PLC) were localized in the plasma membrane of tobacco culture cells. The kinases were significantly inhibited, whereas PLC was markedly activated by 0.1-1 uM Ca^<2+>. A voltage-dependent outward-rectifying Ca^<2+> current was recorded with an outside-out patch of the tonoplast membrane by patch-clamp method. This Ca^<2+> channel was markedly activated by I uM inositol trisphosphate (IP). These results suggest that IP_3, the product of PLC catalysis causes Ca^<2+> release from the vacuole and results in the increase of cytosolic Ca^<2+> co cncentration. The increased Ca^<2+> inhibits the kinases, lowering the supply of substrate to PLC, and results in the decrease in IP_3 production. By such a way, inositolphospholipid metabolism is under the feedback regulation by Ca^<2+>.(2) Ca^<2+>-pump ATPase was solubilized from corn leaf plasma membrane, and separated by an ion exchange HPLC. This was reconstituted in a liposome. The proteoliposome actively took up Ca2 by an ATP-dependent manner, suggesting that the Ca^<2+>-ATPase actually functioned as the Ca^<2+>-pump.(3) Ca^<2+>-dependent protein kinase (CDPK) was purified to 900-fold from a halophilic alga Dunaliella. CDPK was markedly activated by 0.1-1 uM Ca^<2+> and increased its hydrophobicity. The enzyme was soluble in the absense of Ca^<2+>, while bound to the membrane fraction from cells in the presense of Ca^<2+>. A little protein in the soluble fraction was phosphorylated, meanwhile a number of proteins in the membrane fraction were significantly phosphorylated by CDPK. This result suggests that CDPK is activated by the increase of cytoplasmic Ca^<2+> concentration and translocated to the membrane where the substrate proteins are present. The involvement of CDPK in osmoregulation of Dunaliella is expected.

(1)在烟草培养细胞质膜上发现了肌醇磷脂代谢转换酶、磷脂酰肌醇激酶、磷脂酰肌醇单磷酸激酶和磷脂酶C（PLC）。0.1-1 μ M Ca^<2+>可显著抑制激酶活性，而激活PLC活性。采用膜片钳技术，在离体细胞外膜上记录到电压依赖性外向整流Ca^2+电流。1 μ M三磷酸肌醇（IP）可显著激活该Ca^<2+>通道.这些结果表明，PLC催化产物IP_3引起液泡中Ca^2+释放，导致胞浆Ca^2+浓度升高。Ca^2+浓度的增加抑制了这些激酶，降低了PLC的底物供应，导致IP_3的产生减少。通过这种方式，肌醇磷脂代谢处于Ca^2+的反馈调节之下。(2)从玉米叶片质膜中溶解Ca^<2+>-泵ATP酶，并通过离子交换HPLC分离。将其在脂质体中重构。蛋白脂质体以ATP依赖的方式主动摄取Ca 2，表明Ca^2+-ATP酶实际上起着Ca^2+泵的作用。(3)钙离子依赖性蛋白激酶（CDPK）从嗜盐盐杜氏藻中纯化到900倍。0.1-1 μ M Ca^<2+>可显著激活CDPK，并增加其疏水性。该酶在无Ca^<2+>条件下可溶，而在有Ca^<2+>条件下与细胞膜组分结合。可溶性组分中有少量蛋白被磷酸化，而膜组分中有大量蛋白被磷酸化。这一结果表明，胞浆Ca^2+浓度的增加激活了CDPK，并将其转移到存在底物蛋白的细胞膜上。预期CDPK参与杜氏藻的代谢调节。

项目成果

Minobu Kasai: "Reconstitution of Ca^<2+>-pump of plasma membrane from corn leaves" FEBS Lett.

Minobu Kasai：“玉米叶质膜 Ca^2-泵的重建”FEBS Lett。

Minobu Kasai: "Solubilization and reconstitution of Ca^<2+>ーpump from corn leaf plasma membrane" Plant Physiol.

Minobu Kasai：“玉米叶质膜 Ca^2+泵的溶解和重建”植物生理学。

Minobu Kasai: "Solubilization and reconstitution of CaAA2+BBーpump from corn leaf plasma membrane" Plant Physiol.

Minobu Kasai：“玉米叶质膜 CaAA2+BB 泵的溶解和重建”植物生理学。

Yoshiaki Kamada,: "Ca^<2+> regulation of phosphatidylinositol turnover in the plasma membrane from tobacco suspension culture cells" Biochim. Biophys. Acta,.

Yoshiaki Kamada，：“烟草悬浮培养细胞质膜中磷脂酰肌醇周转的Ca ^ 2 调节”Biochim。

Takashi Yuasa: "Ca^<2+>ーdependent protein kinase from <Duanliella>___ー <tertiolecta>___ー"

Takashi Yuasa：“Ca^<2+>ー依赖于 <Duanliella>___ー <tertiolecta>___ー 的蛋白激酶”