V- and P-type Ca2+-stimulated ATPases in a calcifying strain of Pleurochrysis sp. (Haptophyceae)
V- and P-type Ca2+-stimulated ATPases in a calcifying strain of Pleurochrysis sp. (Haptophyceae)
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DOI：
10.1046/j.1529-8817.1998.340079.x
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发表时间：
1998-02-01
影响因子：
2.9
通讯作者：
Gonzalez, EL
中科院分区：
文献类型：
--
作者：
Araki, Y;Gonzalez, EL
颗石藻是海洋单细胞藻类，其特征是能够进行受控的亚细胞钙化。膜结合的Ca ~（2+）刺激的ATP酶的生化和动力学特征进行了研究。用蔗糖密度离心法从侧鞭金藻（Pleurochrysissp.）（CCMP 299）的无菌培养物中分离出膜和细胞器。在叶绿体、高尔基体、质膜和球石囊泡中检测到高水平的Ca ~（2+）刺激的ATP酶。在细胞器和膜中的酶活性的灵敏度进行了评估，已知是特定的几种异构体的钙离子刺激的ATP酶的药理学试剂。Ca 2+刺激的ATP酶活性在高尔基体和球石囊泡制剂是敏感的硝酸盐，硫氰酸盐，叠氮化钠和钒酸盐，环匹阿尼酸，毒胡萝卜素不敏感。ATP依赖的H+运动，但不是Ca-45（2+）的运输，通过球石囊泡被证明。Ca ~（2+）刺激的质膜ATP酶对钒酸敏感。证明了来自梯度分离质膜的微粒体材料的Ca-45（2+）的ATP依赖性、钒酸盐敏感性流出。从分离的高尔基体和球石囊泡制剂的多肽交叉反应的抗体提出对燕麦根质子泵的一个亚基，而从叶绿体制剂的多肽没有交叉反应。这些结果表明，V型Ca ~（2+）激活的ATP酶位于球石囊泡膜上，P型Ca ~（2+）激活的ATP酶位于质膜上。
Coccolithophorids are marine unicellular algae characterized by their ability to carry out controlled, subcellular calcification. The biochemical and kinetic features of membrane-bound Ca2+-stimulated ATPases have been examined. Membranes and organelles from axenic cultures of Pleurochrysis sp. (CCMP299) were isolated by means of sucrose density centrifugation. High levels of Ca2+-stimulated ATPase were detected in chloroplasts, Golgi apparatus, plasma membrane, and coccolith vesicles. The sensitivity of the enzyme activity in the organelles and membranes was assessed with pharmacologic agents that are known to be specific for the several isoforms of Ca2+-stimulated ATPase. The Ca2+-stimulated ATPase activity in the Golgi and coccolith vesicle preparations was sensitive to nitrate, thiocyanate, and sodium azide and insensitive to vanadate, cyclopiazonic acid, and thapsigargin. ATP-dependent H+ movement, but not Ca-45(2+) transport, across the coccolith vesicle was demonstrated. The Ca2+-stimulated ATPase in the plasma membrane preparation was sensitive to vanadate. ATP-dependent, vanadate-sensitive efflux of Ca-45(2+) was demonstrated for microsomal material derived from gradient-isolated plasma membrane. Polypeptides from isolated Golgi and coccolith vesicle preparations cross-reacted to an antibody raised against a subunit of the oat root proton pump, whereas polypeptides from the chloroplast preparations did not cross-react. These findings show that a V-type Ca2+-stimulated ATPase is located on the coccolith vesicle membrane and a P-type Ca2+-stimulated ATPase is located on the plasma membrane.