The in vivo highly tissue-dependent abscisic acid (ABA) specific-binding sites localized in cytosol were identified and characterized in the flesh of developing apple ( Malus pumila L. cv. Starkrimon) fruits. ABA binding activity was scarcely detectable in the microsomes and the cytosolic fraction isolated from the freshly harvested fruits via an in vitro ABA binding incubation of the subcellular fractions. If, however, instead that the subcellular fractions were in vitro incubated in H-ABA binding medium, the flesh tissue discs were directly in vivo incubated in H-ABA binding medium, a high ABA binding activity to the cytosolic fraction isolated from these tissue discs was detected. The in vivo ABA binding capacity of the cytosolic fraction was lost if the tissue discs had been pretreated with boiling water, indicating that the ABA binding needs a living state of tissue. The in vivo tissue-dependent binding sites were shown to possess protein nature with both active serine residua and thiol-group of cysteine residua in their functional binding center. The ABA binding of the in vivo tissue-dependent ABA binding sites to the cytosolic fraction was shown to be saturable, reversible, and of high affinity. The scatchard plotting gave evidence of two different classes of ABA binding proteins, one with a higher affinity ( Kd =2.9 nmol/L) and the other with lower affinity ( Kd =71.4 nmol/L). Phaseic acid, 2- trans -4- trans -ABA or cis-trans -(－)-ABA had substantially no affinity to the binding proteins, indicating their stereo-specificity to bind physiologically active ABA. The time course, pH- and temperature-dependence of the in vivo tissue-dependent binding proteins were determined. It is hypothesized that the detected ABA-binding proteins may be putative ABA-receptors that mediate ABA signals during fruit development.
苹果果实细胞质中依赖活体组织的ABA高亲和力特异结合蛋白
张大鹏* 陈尚武
（中国农业大学果树分子发育实验室，中国农业大学植物生理生化农业部重点开放实验室，北京100094）
摘要：将苹果(Malus pumila L. cv. Starkrimon)果肉微粒体和细胞可溶组分在含有 3H-ABA的缓冲介质中分别温育,仅在细胞可溶组分中测到微弱的3H-ABA结合活性.但是,如果将果肉组织圆片在3H-ABA缓冲介质中直接温育,经制备亚细胞组分后直接测定,在细胞可溶组分中测到很高的3H-ABA特异结合活性.果肉圆片用沸水预先热处理使细胞可溶组分中的3H-ABA结合活性完全丧失,说明ABA结合依赖于组织的活体状态 .药理实验证明了ABA结合位点的蛋白质性质,同时证明该蛋白的活性中心具有-SH和丝氨酸基团.ABA结合蛋白对ABA的结合具有可饱和性、可逆性和高亲和力.Scatchard作图证明存在2种ABA结合蛋白,一种具有较高的亲和力,其解离常数(Kd)为2.9 nmol/L,另一种亲和力相对较低,其Kd值为71.4 nmol/L.用ABA结构相似物进行的竞争实验证明了AB A结合蛋白对配体结合的立体特异性.分析了ABA结合蛋白与ABA结合的时间曲线、pH和温度依赖性. 本研究检测到的依赖活体组织的ABA结合蛋白可能是果实发育过程中介导ABA信号的受体.
关键词： 脱落酸；脱落酸结合蛋白；苹果果实
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