玉米根ABA结合蛋白的亚细胞定位及动力学性质

以玉米（Ｚｅａ ｍａｙｓＬ．）根或胚芽鞘为材料，经匀浆、分级离心得到胞质部分和膜部分（微粒体），进一步用６．２％ （Ｗ／Ｗ ） Ｄｅｘｔｒａｎ Ｔ５００ 和ＰＥＧ ３３５０ 两相系统制备质膜，用１％ 和８％ （Ｗ ／Ｗ） Ｄｅｘｔｒａｎ Ｔ７０ 梯度离心制备液泡膜． 电镜鉴定及多种标志酶检测表明，制备获得了高纯度正向型质膜和富含液泡膜的组分，其它内膜的污染很少． 用微量放射配体结合（ＭＲＬＢ）实验证明，玉米根微粒体的ＡＢＡ专一性结合位点主要分布在液泡膜和质膜上，这两种膜组分与ＡＢＡ 的特异结合活性分别为２４８５．４ ｆｍ ｏｌ／ｍ ｇ ｐｒｏｔｅｉｎ 和１２５７．３ ｆｍ ｏｌ／ｍ ｇ ｐｒｏ－ｔｅｉｎ，玉米根段胞质部分结合活性最低（差一个数量级）．质膜上ＡＢＡ－ＢＰ与ＡＢＡ 的结合平衡解离常数（ＫＤ）为１．５７ ｎｍ ｏｌ／Ｌ．

SUBCELLULAR LOCALIZATION AND KINETIC CHARACTERIZATION OF ABA BINDING PROTEINS IN MAIZE ROOT

By means of differential centrifugation, cytosol fraction and microsome were prepared from maize roots which have been grown in dark for 4 d.Highly purified plasma membranes were isolated from the microsome in two phase aqueous system which is composed of 6 2% (W/W) Dextran T500 and PEG 3350. The tonoplast was collected from the interface between 1% and 8% (W/W) Dextran T70 after gradient centrifugation.Electron microscopic observation and marker enzyme activities analysis proved that these fractions contained very few other membranes. Microvolume radioactivity ligand binding assay indicated that the specific binding sites of ABA in maize root microsome were mainly distributed on tonoplast and plasma membrane fractions. Their specific binding activity was 2485 4 and 1257 3 fmol/mg protein, respectively, the specific binding activity of cytosol fraction being the lowest (one order of magnitude lower). The dissociation constant (K D) of ABA BP in plasma membrane was 1 57 nmol/L.