Intracellular Calcium Ion Signal Transduction System in Plant

(1)烟草培养细胞质膜上存在与肌醇磷脂、磷脂酰肌醇激酶、磷脂酰肌醇一磷酸激酶和磷脂酶C代谢代谢有关的酶。0.1-1umCa~(2+)和Gt~(2+)可显著抑制PLC的活性，而对PLC有明显的激活作用。用膜片钳技术在液泡膜上由外向外记录电压依赖性外向整流钙电流。该钙通道可被肌醇三磷酸(IP)显著激活。这些结果表明，PLC催化产物IP_3引起液泡内Ca~(2+)的释放，导致胞内Ca~(2+)浓度升高。Ca~(2+)~(2+)和Gt~(2+)的增加抑制了该酶的活性，减少了对PLC的底物供应，导致了IP_3产量的下降。通过这种方式，肌醇磷脂代谢受到Ca~(2+)&gt；的反馈调节。(2)从玉米叶片质膜中溶解Ca~(2+)-Pump ATPase，并用离子交换高效液相色谱分离。这是在脂质体中重组的。该蛋白脂体以一种依赖于ATP的方式主动摄取Ca~(2+)，表明Ca~(2+)-ATPase实际上起着Ca~(2+)-泵的作用。(3)从一种嗜盐藻类杜氏藻中分离纯化了约900倍的依赖于Ca~(2+)的蛋白激酶(CDPK)。0.1-1um Ca~(2+)和Gt~(2+)可显著激活CDPK，并使其疏水性增强。该酶不含Ca~(2+)和Gt~(2+)，但以Ca~(2+)和Gt~(2+)形式结合在细胞膜上。可溶性组分中的少量蛋白质被CDPK磷酸化，而膜组分中的许多蛋白质被CDPK显著磷酸化。这一结果表明，CDPK被胞质内Ca~(2+)和Gt~(2+)浓度的升高激活，并被转移到底物蛋白所在的膜上。CDPK可能参与了杜氏杆菌的渗透调节。

(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.