For understanding the function of tonoplast protein in plant cell signal pathway, we have identified an integral protein kinase activity from the highly purified tonoplast isolated from maize (Zea mays L.) root by a new nonradioactive method in which a color labeled peptide was used as substrate. The protein kinase was Ca2+ dependent and CaM and phosphatidylserine independent, like the calmodulin like domain protein kinase (CDPK) in many plants. The optimal pH value and Ca2+ concentration were 6.5 and 10 μmol/L, respectively. According to the optimal pH value and the effect of detergent, it could be inferred that the active site of this protein kinase is oriented toward the cytoplasm. Zn2+ had no obvious effect on its activity, indicating that this protein kinase has no zinc finger domain that exists in some mammalian protein kinases. At the same time, when tonoplast proteins were prephosphorylated in the presence of Ca2+ and ATP, both the ATP hydrolysis and the proton transport activity of vacuolar H+ ATPase were stimulated. This stimulation could be reversed by an alkaline phosphatase. These results indicate that a Ca2+ dependent protein kinase was located in the tonoplast, and a Ca2+ dependent phosphorylation, probably caused by this kinase, activated the vacuolar H+ ATPase activity. These results are helpful for further research on the function of CDPK in the course of signal transduction in plants.
玉米根尖细胞液泡膜结合的蛋白激酶的存在及其性质
陈 硕 陈 珈* 王学臣
（中国农业大学生物学院，植物生理生化国家重点实验室，北京100094）
摘要: 为了解液泡膜蛋白在植物细胞信号途径中的功能,用新型的非放射性同位素方法从玉米根细胞的高纯度液泡膜上鉴定出一种膜内在的蛋白激酶.这种蛋白激酶具有Ca 2+依赖、CaM和磷脂酰丝氨酸不依赖等特性,与已在多种植物中报道的含有类似钙调素结构域的蛋白激酶CDPK相似.离体实验表明其活性的最适pH值为6.5,最适Ca 2+浓度为10 μmol/L.从最适pH值和去污剂的影响可以推测出其活性位点朝向胞质一侧.Zn 2+对其活性没有明显的抑制作用,说明该激酶缺少某些哺乳动物的蛋白激酶常含有的锌指结构.当液泡膜蛋白在Ca 2+和ATP存在的条件下被预磷酸化后,液泡膜H +-ATPase的ATP水解和质子转运过程均被激活.激活的活性可以被碱性磷酸酶逆转.以上结果说明玉米根尖细胞的液泡膜中存在一种可能是CDPK的蛋白激酶.由它造成的Ca 2+依赖的磷酸化作用激活了液泡膜H+-ATPase的活性.这些结果将有助于深入研究CDPK在植物细胞信号转导中的功能.
关键词： 蛋白激酶；磷酸化H+-ATPase液泡膜；玉米
通讯作者。E-mail:<chenja @ public.bta.net.cn>.
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