Intracellular Calcium Ion Signal Transduction System in Plant

植物细胞内钙离子信号转导系统

基本信息

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

项目摘要

(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）在烟草膜的肿瘤膜中定位，将参与肌硝基磷脂，磷脂酰肌醇激酶，磷脂酰肌醇 - 磷酸激酶激酶和磷脂酶C（PLC）的酶进行。激酶被显着抑制，而PLC被0.1-1 UM Ca^<2+>显着激活。通过贴片钳方法记录了用托管膜的外部斑块记录电压依赖的向外矫正CA^<2+>电流。该CA^<2+>通道被I UM肌醇三磷酸盐（IP）显着激活。这些结果表明，PLC催化的乘积IP_3导致Ca^<2+>从液泡中释放，并导致胞质Ca^<2+> Co Cncentration的增加。增加的Ca^<2+>抑制了激酶，降低了对PLC的底物的供应，并导致IP_3产生的减少。通过这种方式，印辛磷脂代谢在反馈调节下通过Ca^<2+>。（2）Ca^<2+> - 泵ATPase从玉米叶质膜中溶解，并通过离子交换HPLC分离。这是在脂质体中重构的。蛋白脂质体通过ATP依赖性的方式积极地摄入Ca2，这表明Ca^<2+> - ATPase实际上用作Ca^<2+> - 泵。（3）Ca^<2+> - 依赖性蛋白激酶（CDPK）从卤代alga dunaliella纯化为900倍。 CDPK被0.1-1 UM CA^<2+>显着激活，并增加了其疏水性。该酶可溶于不存在Ca^<2+>，而与Ca^<2+>的细胞的膜分数结合。可溶性级分中的一点蛋白质被磷酸化，与此同时，膜级分中许多蛋白质被CDPK显着磷酸化。该结果表明，CDPK通过胞质Ca^<2+>浓度的增加而激活CDPK，并易位到存在底物蛋白的膜。预计CDPK参与了Dunaliella的渗透调节。