葡聚糖与蔗糖密度梯度离心制备膜微囊方法的比较

COMPARISON OF DEXTRAN T70 AND SUCROSE DENSITY GRADIENTS CENTRIFUGATION FOR PREPARING MEMBRANE VESICLES

分别以不连续蔗糖［２２０／３６０／４５０（ｇ／Ｌ）］和葡聚糖（ｄｅｘｔｒａｎＴ７０）［６０／１２０（ｇ／Ｌ）］两种介质密度梯度，制备并比较了大麦幼根细胞质膜和液泡膜微囊。结果表明，两种介质梯度制备的质膜微囊纯度较高，无线粒体膜和液泡膜污染；液泡膜微囊虽基本上去除了线粒体膜，但均混有少量质膜碎片。用蔗糖梯度制备的质膜微囊较葡聚糖梯度制备的质膜微囊有较高的翻转比率；对液泡膜微囊，前者的原位比例也较后者略高。另外，液泡膜Ｈ＋－ＰＰａｓｅ活性测定反应体系中应加入钼酸钠抑制剂，反应终止液和显色液可与Ｈ＋－ＡＴ－Ｐａｓｅ相同。因此，在制备质膜和液泡膜进行Ｈ＋－ＡＴＰａｓｅ或Ｈ＋－ＰＰａｓｅ生化特性的研究时，可用蔗糖代替葡聚糖。

The plasma membrane and tonoplast vesicles from the roots of barley seedling were prepared and compared by discontinuous sucrose and dextran T70 density gradients centrifugation. The results showed that the plasma membrane vesicles prepared by two kinds of density gradients had high purity and were not polluted by mitochondria and tonoplast, the tonoplast vesicles, although with no mitochondria, were adulterated by a little plasma membrane vesicles. The percentage of insideout vesicles of plasma membrane prepared by sucrose density was higher than that of plasma membrane prepared by dextran T70. The percentage of rightsideout vesicles of tonoplast prepared by sucrose density was also slightly higher than that of tonoplast prepared by dextran T70. For to determine the activity of tonoplast H+PPase, Namolybdate inhibitor should be added to the reaction mixture for the accuracy, and the solutions for terminating and colouring may be the same as H+ATPase. From the mentioned above, to prepare the plasma membrane and tonoplast vesicles for the studies on H+ATPase or H+PPase, the sucrose density can take the place of dextran T70.