Plant Cell Biology Group, Research School of Biological Sciences, The Australian National University, ACT 2601, Canberra, Australia.
Planta. 1992 Aug;188(1):54-61. doi: 10.1007/BF00198939.
Cytoplasmic streaming in the characean algae is inhibited by micromolar rises in the level of cytosolic free Ca(2+), but both the mechanism of action and the molecular components involved in this process are unknown. We have used monoclonal antibodies against soybean Ca(2+)-dependent protein kinase (CDPK), a kinase that is activated by micromolar Ca(2+) and co-localizes with actin filaments in higher-plant cells (Putnam-Evans et al., 1989, Cell Motil. Cytoskel. 12, 12-22) to identify and localize its characean homologue. Immunoblot analysis revealed that CDPK in Chara corralina Klein ex. Wild shares the same relative molecular mass (51-55 kDa) as the kinase purified from soybean, and after electrophoresis in denaturing gels is capable of phosphorylating histone III-S in a Ca(2+)-dependent manner. Immunofluorescence microscopy localized CDPK in Chara to the subcortical actin bundles and the surface of small organelles and other membrane components of the streaming endoplasm. The endoplasmic sites carrying CDPK were extracted from internodal cells by vacuolar perfusion with 1 mM ATP or 10(-4) M Ca(2+). Both the localization of CDPK and its extraction from internodal cells by perfusion with ATP or high Ca(2+) are properties similar to that reported for the heavy chain of myosin in Chara (Grolig et al., 1988, Eur. J. Cell Biol. 47, 22-31). Based on its endoplasmic location and inferred enzymatic properties, we suggest that CDPK may be a putative element of the signal-transduction pathway that mediates the rapid Ca(2+)-induced inhibition of streaming that occurs in the characean algae.
在石莼藻类中，细胞质的流动会被细胞溶质游离 Ca(2+) 水平的微摩尔上升所抑制，但该过程的作用机制和涉及的分子成分尚不清楚。我们使用针对大豆 Ca(2+) 依赖性蛋白激酶 (CDPK) 的单克隆抗体，该激酶在微摩尔 Ca(2+) 作用下被激活，并在高等植物细胞中与肌动蛋白丝共定位（Putnam-Evans 等人，1989 年，细胞运动。细胞骨架。12，12-22），以鉴定和定位其 characean 同源物。免疫印迹分析表明，Chara corralina Klein ex. Wild 中的 CDPK 与从大豆中纯化的激酶具有相同的相对分子质量（51-55 kDa），并且在变性凝胶电泳后能够以 Ca(2+) 依赖的方式磷酸化组蛋白 III-S。免疫荧光显微镜将 CDPK 定位在石莼的皮层下肌动蛋白束和小细胞器以及流动内质的表面。通过用 1 mM ATP 或 10(-4) M Ca(2+) 进行液泡灌注从节间细胞中提取带有 CDPK 的内质。CDPK 的定位及其通过 ATP 或高 Ca(2+) 灌注从节间细胞中提取，均具有与石莼中肌球蛋白重链报告的特性相似（Grolig 等人，1988 年，欧洲细胞生物学杂志。47，22-31）。基于其内质定位和推断的酶学特性，我们认为 CDPK 可能是介导石莼藻类中快速 Ca(2+) 诱导的流动抑制的信号转导途径的一个假定元件。
