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Calcium-dependent protein kinase in osmoregulation of the halotolerant green alga Dunaliella tertiolecta

钙依赖性蛋白激酶在耐盐绿藻 Dunaliella tertiolecta 渗透调节中的作用

基本信息

批准号：
06454013
负责人：
TORIYAMA Shoshi
金额：
$ 4.29万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

Calcium-dependent protein kinase (CDPK)was purified 20,000-fold from soluble franction of Dunaliella, and characterized. Using an anti-Dunaliella CDPK antibody, cDANs encoding parts of CDPK were cloned from a Dunaliella cDNA library. A full length cDNA was isolated by using one of partial cDNAs as a probe, and is nucleotide sequence was determined. Deduced amino acid squence from the nucleotide sequence indicated that Dunaliella CDPK in a typical CDPK which has kinase domain at N-terminus, calmolulin-like domain at C-terminus and pseudosubstrate domain between them.When subjected to hypo-/hypersomotic shock, 40-kDa protein kinases named HAP/LAP kinases, respectively, which have different substrate specificities and time courses of activation were activated. The activation accompanied with protein phosphorylation. Since an involvement of increase in cytosolic Ca^<2+> was anticipated for the activation, tobacco suspension culture cells transformed aequorin was subjected osmotic shock. Upon hypoosmotic shock, a transient increase in Ca^<2+> with a peak at 70 sec after the shock and subsequent activation of 50-, 75- and 80-kDa protein kinases was observed. The activation accompanied with protein phosphorylation suggesting the involvement of CDPK.A CDPK cross-reacting with anti-Dunaliella CDPK was detected in a brackish water Characeae Lamprothamnium. When this alga injected the antibody into the cytoplasm was subjected to hypoosmotic shock, turgor regulation was suppressed even though an increase in cytosolic Ca^<2+> occured after the shock. This result suggests the involvement of CDPK in turgor regulation of this alga.The results obtained in this study suggest that CDPK is tightly involved in osmotic signal transduction and osmoregulation in algae and higher plants.

钙依赖性蛋白激酶（CDPK）纯化20,000倍，从杜氏藻的可溶性果糖浆，并进行了表征。使用抗杜氏盐藻CDPK抗体，从杜氏盐藻cDNA文库中克隆编码CDPK部分的cDNA。以部分cDNA为探针，分离得到全长cDNA，并测定其核苷酸序列。由核苷酸序列推导的氨基酸序列表明，杜氏盐藻CDPK为典型的CDPK，其N端为激酶结构域，C端为钙调蛋白样结构域，中间为假底物结构域，当受到低/高休克时，40 kDa的蛋白激酶HAP/HAP激酶分别被激活，这两种蛋白激酶具有不同的底物特异性和激活时程。这种激活伴随着蛋白质磷酸化。由于预期激活涉及胞质Ca^2+的增加，因此对转化水母发光蛋白的烟草悬浮培养细胞进行渗透压休克。在低渗休克时，观察到Ca^<2+>短暂升高，在休克后70秒达到峰值，随后50、75和80 kDa蛋白激酶被激活。在微咸水藻中检测到一种CDPK与抗杜氏藻CDPK的交叉反应。当将抗体注射到细胞质中的小鼠受到低渗休克时，即使休克后胞浆Ca^<2+>增加，膨压调节也受到抑制。这一结果表明，CDPK参与膨压调节。在本研究中获得的结果表明，CDPK是密切参与渗透信号转导和藻类和高等植物的膨压调节。