胡杨液泡膜微囊的纯化及其质子转运活性

 为进一步研究液泡膜及 H+ - ATP酶在胡杨抵御盐胁迫中所起的作用 ,比较了研磨、捣碎和超声破碎三种细胞破碎方法 ,从悬浮培养的胡杨细胞中制备液泡膜微囊的效果 ;并用差速离心和不连续蔗糖密度梯度离心纯化了胡杨液泡膜微囊 .通过测定 H+ - ATP酶对 NO-3 、VO3-4和 Na N3的敏感性 ,以及焦磷酸酶质子转运活性表明 ,液泡膜微囊主要分布在 0 %～ 2 5%的蔗糖界面上 .捣碎法破碎细胞结合差速离心和蔗糖密度梯度离心可获得正向微囊比例高、封闭性好和酶活性高的液泡膜微囊

Purification of Tonoplast Vescles from Populus euphratica and Its H~+-Pumping Activity

Through differential centrifugation and sucrose density gradient centrifugation,tonoplast vesicles were isolated from suspension cultured Populus euphratica cells broken by mortar and pestle,blender or ultrasonic homogenizer.The sensitivities of H + ATPase to NO - 3,VO 3- 4 and NaN 3,as well proton pumping activities of membrane bound pyrophosphatases demonstrated that tonoplast vesicles mainly distributed on the interface of 0%～25% of sucrose.The effect of various cell breaking methods on H + ATPase activities,the orientation of H + ATPase in the vesicles and protein recovery were analyzed.It was found that the tonoplast vesicles purified from cells broken by blender were intact,possessed higher ratio of right side out vesicles and had higher enzyme activity.