两相法分离纯化橡胶树树皮质膜

Separation and purification of plasma membrane from bark of Hevea brasiliensis by aqueous two-phase partition

橡胶树树皮质膜H^+-ATPase在橡胶树产排胶过程中扮演着重要角色,制备高纯度及高活性的质膜是研究质膜H^+-ATPase特性和功能的必要条件。该研究以一年生巴西橡胶树(Hevea brasiliensis)树皮为材料,利用差速离心法获得粗膜微粒体,通过两相分配法分离纯化质膜,并研究两相体系中不同浓度聚合物(5.9%、6.1%、6.3%、6.5%、6.7%,W/W)和KCl(2、5、8、11、14 mmol·L^(-1))对质膜蛋白得率和纯化效率的影响。通过Bradford法对质膜蛋白得率进行检测,同时采用酶活性检测法对质膜纯度进行检测,分析结果表明选用6.4%(W/W)聚合物浓度和5mmol·L^(-1)KCl组成的两相体系可获得较高纯度和得率的橡胶树树皮质膜。通过电镜观察法在形态学上对质膜纯度进一步评价,利用铅铀能侵染全部膜组分使其染色,而磷钨酸只能专一性地侵染质膜并使其染色这一特性,分别使用铅铀和磷钨酸对切片进行染色,并通过透射电镜对切片染色程度进行直接观察,结果表明提取的粗膜微粒体中质膜组分较少,存在大量的细胞器膜污染,而纯化后的质膜膜组分较单一,其他膜组分污染较少,而且质膜大小较均一,可以用于进行后续橡胶树树皮质膜H^+-ATPase特性和功能的研究。

The plasma membrane H^＋-ATPase of rubber tree bark involved in various important physiological processes,such as ion transportation,stress responses,production and release of natural rubber latex,the preparation of a high purity and activity plasma membrane is the first and essential step to understand the functions of H^＋-ATPase in some in situ situations. In the present study,the separation of crude microsomes was isolated by differential and gradient centrifugations,the aqueous two-phase partition system was employed to separate the organelles membrane constitutions and receive highly pure plasma membrane,and the effect on the extraction efficiency of polymer and KCl concentration in partition was studied as considered the characteristic of bark of rubber tree. The total concentration of plasma membrane proteins was measured by Bradford method. The purity of different membrane was determined by the sensitivity of representative enzyme activity measured by Molybdenum blue method. H^＋-ATPase of plasma membrane was specifically inhibited by Na VO3 under p H 6.5,due to different types of H^＋-ATPase in different organelle membranes,while H^＋-ATPase of tonoplast or plastid membrane was suppressed by KNO3 or Na N3 under pH 8.5,respectively. Our results indicated that the combination of 6.4% （W/W） polymer and 5 mmol·L^-1KCl applied in two-phase partition system achieved the relatively high purity and yield ratio of plasma membrane whereas high polymer concentration of or KCl could increase the purity but with poor productivity. In order to further verify that morphological diversity between purified plasma membrane and crude microsomes,transmission electron microscopy （TEM） was performed. Paraffin embedded thin sections was made and stained separately with lead uranium and phosphotungstic acid. The morphological analysis showed that in crude microsomes there was little plasma membrane and full of cross-contaminant membrane substitutions,while after purified,the constitution of plasma membrane was relatively pure with less contaminant. In summary,highly purified plasma membrane was yielded by the aqueous two-phase partition system with 6.4% （W/W） polymer and 5 mmol·L^-1KCl and perform a comparatively singular morphology,which could be further used to reveal the function of H^＋-ATPase of bark of rubber tree in production and release of rubber and other physiological processes.