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以玉米(Zea mays L.)根为材料,采用分级离心和两相分配法制备高纯度的质膜。实验比较了Tri-ton X-100和丙酮两种增溶剂对膜上ABA结合蛋白(ABA-BPm)的增溶效果。结果表明0.2％(W/V) TritonX-100的增溶效率超过85％,而丙酮法增溶效率仅达65％。放射配基(~3H-ABA)结合分析表明,增溶的膜蛋白与ABA的结合反应具有竞争性、饱和性、专一性和高亲和性等特点,而相同反应条件下的BSA则没有这些特征,证明了质膜上存在着ABA结合蛋白。实验还比较了ABA-BPm与增溶的ABA结合蛋白(ABA-BPs)与ABA的特异结合活性,结果显示ABA-BPs对反应温度和介质pH更为敏感,保持最大结合活性的时间也较短(<30min),暗示着增溶膜蛋白离开膜脂环境后更不稳定、易失活。
Using corn (Zea mays L.) as material, high purity plasma membrane was prepared by fractionation centrifugation and two-phase partitioning. The solubilization of ABA-binding protein (ABA-BPm) by Tri-ton X-100 and acetone was compared. The results showed that the TritonX-100 0.2% (W / V) solubilization efficiency of more than 85%, while acetone solubilization efficiency of only 65%. Radioligand (~ 3H-ABA) binding analysis showed that the binding reaction of solubilized membrane protein and ABA was competitive, saturated, specific and high affinity, while the BSA under the same reaction conditions did not These features demonstrate the presence of an ABA-binding protein on the plasma membrane. The specific binding activity of ABA-BPs to ABA was also compared between ABA-BPm and ABA-ABs. The results showed that ABA-BPs were more sensitive to reaction temperature and media pH, and the retention time of maximal binding activity was also shorter (<30min), suggesting that the solubilized membrane protein leaves the membrane lipid environment more unstable and easily inactivated.