Canut H, Brightman A, Boudet A M, Morré D J
Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907.
Plant Physiol. 1988 Feb;86(2):631-7. doi: 10.1104/pp.86.2.631.
Absolute orientations (sidedness) of plasma membrane vesicles obtained in highly purified fractions by preparative free-flow electrophoresis and by aqueous two-phase partition were determined based on ATPase latency and morphological criteria. Free-flow electrophoresis yielded two plasma membrane fractions. One, the least electronegative and designated fraction ;E,' was pure plasma membrane. The other, more electronegative and designated fraction ;C,' was heavily contaminated by various other cellular membranes. Plasma membrane vesicles from both fraction C and fraction E partitioned into the upper phase with aqueous two-phase partitioning. Purified plasma membrane obtained from microsomes by two-phase partition (upper phase) when subjected to free-flow electrophoresis also yielded two fractions, one fraction co-migrated with fraction C and another fraction co-migrated with fraction E. Both fractions exhibited an ATPase activity sensitive to vanadate and insensitive to nitrate and azide. ATPase activity was used as a structure-linked latency marker for the inner membrane surface. Concanavalin A binding (linked to peroxidase) was used as an imposed electron microscope marker for the outer membrane surface. Fraction E vesicles showed low ATPase latency (two-fold or less) and weak reactivity with concanavalin A peroxidase. In contrast, fraction C vesicles were characterized by much greater latencies upon detergent treatment (sevenfold) and a strong reaction with concanavalin A peroxidase. Two-phase partition as the initial procedure for plasma membrane isolation, yielded mixtures of vesicles of both inside out and right-side out orientation. Free-flow electrophoresis resolved the plasma membrane isolates into vesicles from fraction C which were right-side out (cytoplasmic side in), and vesicles from fraction E which were wrong-side out (cytoplasmic side out). Therefore, the two methods used in series, provided highly purified membrane preparations of apparently homogenous vesicles of opposite known absolute orientations.
基于ATP酶潜伏性和形态学标准，确定了通过制备型自由流电泳和水相两相分配法在高度纯化的组分中获得的质膜囊泡的绝对取向（方向性）。自由流电泳产生了两个质膜组分。其中一个，电负性最小且指定为组分“E”，是纯质膜。另一个，电负性更大且指定为组分“C”，被各种其他细胞膜严重污染。来自组分C和组分E的质膜囊泡在水相两相分配中都分配到上相中。通过两相分配（上相）从微粒体中获得的纯化质膜在进行自由流电泳时也产生了两个组分，一个组分与组分C共同迁移，另一个组分与组分E共同迁移。两个组分都表现出对钒酸盐敏感而对硝酸盐和叠氮化物不敏感的ATP酶活性。ATP酶活性被用作内膜表面的结构相关潜伏性标记。伴刀豆球蛋白A结合（与过氧化物酶相连）被用作外膜表面的外加电子显微镜标记。组分E囊泡显示出低ATP酶潜伏性（两倍或更低）且与伴刀豆球蛋白A过氧化物酶的反应较弱。相比之下，组分C囊泡的特征是在去污剂处理后潜伏性大得多（七倍）且与伴刀豆球蛋白A过氧化物酶有强烈反应。两相分配作为质膜分离的初始步骤，产生了内膜外翻和外膜外翻取向的囊泡混合物。自由流电泳将质膜分离物解析为来自组分C的外膜外翻（细胞质面朝内）囊泡和来自组分E的内膜外翻（细胞质面朝外）囊泡。因此，串联使用的这两种方法提供了具有明显相反已知绝对取向的、高度纯化的、明显均匀的囊泡膜制剂。
Zotero 插件
