水稻叶片Ca²⁺/CaM依赖性蛋白激酶的纯化和活性测定

水稻叶片Ca 2+ /CaM依赖性蛋白激酶的纯化和活性测定 Purification and Activity Determination of a Ca 2+ /CaM-Dependent Protein Kinase from Leaves Rice 胡德文 HU De\|wen 1 first-author 何之常 HE Zhi\|chang 1 史红梅 SHI Hong\|mei 1 张景昱 ZHANG Jing\|yu 1 袁文静 YUAN Wen\|jing 1 武汉大学植物发育生物学教育部重点实验室,湖北,武汉,430072 武汉大学植物发育生物学教育部重点实验室,湖北,武汉,430072 Key Laboratory of MOE for Plant Developmental Biology,Wuhan University Key Laboratory of MOE for Plant Developmental Biology,Wuhan University 通过高速离心、（ NH4） 2 SO4沉淀、琼脂糖凝胶过滤和 Ca M- sepharose4B亲和层析 ,从 HPGMR（ 5 8S）叶片中初步纯化出 Ca2 + / Ca M依赖性蛋白激酶 .纯化的 Ca2 + / Ca M依赖性蛋白激酶经 SDS-聚丙烯酰胺凝胶线性梯度电泳 ,亚基分子量约为 5 5× 10 3.在 Ca2 + / Ca M依赖性蛋白激酶的标准反应体系中 ,存在过量的 ATP,提取磷酸化反应剩余的 ATP,用叶绿体 Mg2 + - ATP酶分解 ATP,最后用复合孔雀绿测定无机磷 ,从而来计算 Ca2 + / Ca M依赖性蛋白激酶的活性 .实验结果表明 ,该酶活性是依赖 Ca2 + 和 Ca M的 ,并且 EGTA和 TFP对酶活性有明显的抑制作用 Using ammonium sulfate fraction ,sephorose CL 4B gel filtration and CaM sephorose 4B column Chromatography,a Ca 2+ /CaM PK was purified from leaves of HPGMR 58S Oryza sativa L.subsp japonica. The purified enzyme was electrophoryed by gradient SDS PAGE .The subunit molecular weight is about 55×10 3. The enzyme activity assaying system is excessive ATP in standard reaction system.The surplus ATP of phosphorylation reaction was extracted then was decomposed by chloroplast Mg 2+ ATPase. After that the inorganic phosphorus was assayed by combined malachite green reagent.The research showed that the enzyme activities are stimulated by Ca 2+ and CaM but are depressed by EGTA and TFP. Ca2+/CaM依赖性蛋白激酶 Mg2+-ATP酶 光敏核不育水稻农垦58S Ca 2+ /CaM PK Mg 2+ ATPase HPGMR（58S） 10.14188/j.1671-8836.2000.06.024 Q946.5 国家自然科学基金资助项目 !（ 3 93 70 42 7） 2000-06-01 2021-04-01 6