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.

依赖钙依赖性蛋白激酶（CDPK）从Dunaliella的可溶性填料中纯化了20,000倍，并进行了表征。使用抗Dunaliella cDPK抗体，从dunaliella cDNA库中克隆了编码CDPK部分的CDAN。通过使用部分cDNA作为探针分离全长cDNA，并确定为核苷酸序列。从核苷酸序列中推导的氨基酸挤压表明，典型的CDPK中的dunaliella cdpk在N末端具有激酶结构域，在c末端和假性叠层的结构域，它们之间的钙化蛋白样结构域和它们之间的假卵形结构域。激活的特异性和时间过程被激活。伴随蛋白质磷酸化的激活。由于预计激活胞质Ca^<2+>的增加，因此烟草悬浮培养细胞转化为渗透性的渗透性休克。在低渗透性休克后，观察到休克和随后激活50，75和80-kDa蛋白激酶的Ca^<2+>的短暂增加，在70秒处达到峰值。伴有蛋白质磷酸化的激活表明CDPK的参与。CDPK交叉反应与抗Dunaliella cdpk的含量在微咸水角色Lamprothamnium中检测到。当该藻类将抗体注射到细胞质中时，即使休克后发生胞质Ca^<2+>的增加，也会抑制turgor调节。该结果表明，CDPK参与了该藻类的Turgor调节。在这项研究中获得的结果表明，CDPK与藻类和较高植物的渗透信号转导和渗透调节密切相关。