Intracellular Calcium Ion Signal Transduction System in Plant

(1)烟草培养细胞质膜中存在肌醇磷脂、磷脂酰肌醇激酶、磷脂酰肌醇-单磷酸激酶和磷脂酶C （PLC）等代谢转化酶。0.1-1 μ m Ca^<2+>可显著激活PLC。用膜片钳法在细胞膜外接膜片上记录了电压依赖性外整流Ca^<2+>电流。这个Ca^<2+>通道被I - uM肌醇三磷酸（IP）显著激活。这些结果表明，PLC催化产物IP_3使Ca^<2+>从液泡中释放出来，导致胞质Ca^<2+> co浓度升高。Ca^<2+>的增加抑制了激酶，减少了对PLC的底物供应，导致IP_3的产生减少。这样，肌糖磷脂代谢受到Ca^<2+>的反馈调节。(2)从玉米叶片质膜中溶解Ca^<2+>-泵三磷酸腺苷酶，并用离子交换高效液相色谱法分离。这是在脂质体中重建的。蛋白脂质体通过atp依赖的方式积极吸收Ca2，这表明Ca^<2+>- atp酶实际上起着Ca^<2+>-泵的作用。(3)从嗜盐海藻杜氏藻中纯化Ca^<2+>-依赖性蛋白激酶（CDPK）至900倍。0.1-1 uM Ca^<2+>对CDPK有明显的活化作用，其疏水性增强。该酶在没有Ca^<2+>的情况下可溶，而在Ca^<2+>存在的情况下与细胞的膜组分结合。可溶性部分蛋白被少量磷酸化，膜部分蛋白被CDPK显著磷酸化。这一结果表明，CDPK通过细胞质Ca^<2+>浓度的增加而被激活，并转移到底物蛋白存在的膜上。CDPK参与杜氏藻的渗透调节是有预期的。

(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.