玉米根细胞正面向外和内翻外质膜囊泡的分离

The Isolation of Right-Side-Out and Inside-Out Plasma Membrane Vesicles from Maize Root Cells

利用水双相分配分离法从玉米根细胞提取原生质膜囊泡。其质膜成分特异的K^+,Mg^(2+)—ATP酶活性比膜粗提液高1.67倍。其他细胞器标记酶活性在质膜组分中很低或没有。特异染色电镜形态学定量统计表明质膜组分中90％以上是质膜囊泡。去污剂刺激ATP酶活性实验证明,约92％囊泡是正面向外的。应用此法也证明NAD(P)H—氧化酶可能是跨膜存在的,其催化Fe^(3+)还原的位点可能在膜的内侧。应用高盐冻—融法并结合水双相分配分离法处理正面向外囊泡,可获得内翻外质膜囊泡,实验证明它不仅纯度高而且封闭。

Plasma membranes (PM) from maize (Zeamays) are isolated by aqueoustwo phase partitioning. The isolated membrane fraction shows a 1.67-foldenrichment in activity of Mg, K-ATPase over a microsomal pellet. Activitiesof marker enzyme for other organelles are low or not detectable in PMfraction. Quantitative morphometric analysis using electron microscope indi-cates that PM vesicles are more than 90%. Detergent simulation of ATPaseactivity shows that greater than 92% of PM vesicles are oriented withright-side-out (Cytoplasmic surface inside). By using the same method, itis shown that NAD(P)H-dependeat oxidase is likely to exist across theplasma membrane, and that PM associated with NAD(P)H oxidation orferricyanide reduction is catalysed on sites not exposed to the outer surfaceof the membrane. By freezing and thawing, then separated by repeating thephase partition step, inside-out PM vesicles are obtained. It is ahown thatthe inside-out PM vesicles are not only pure, but also tightly sealed.