Sandelius A S, Penel C, Auderset G, Brightman A, Millard M, Morré D J
Department of Biological Sciences and Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907.
Plant Physiol. 1986 May;81(1):177-85. doi: 10.1104/pp.81.1.177.
A procedure is described whereby highly purified fractions of plasma membrane and tonoplast were isolated from hypocotyls of dark-grown soybean (Glycine max L. var Wayne) by the technique of preparative free-flow electrophoresis. Fractions migrating the slowest toward the anode were enriched in thick (10 nanometers) membranes identified as plasma membranes based on ability to bind N-1-naphthylphthalamic acid (NPA), glucan synthetase-II, and K(+)-stimulated, vanadate-inhibited Mg(2+) ATPase, reaction with phosphotungstic acid at low pH on electron microscope sections, and morphological evaluations. Fractions migrating farthest toward the anode (farthest from the point of sample injection) were enriched in membrane vesicles with thick (7-9 nanometers) membranes that did not stain with phosphotungstic acid at low pH, contained a nitrate-inhibited, Cl-stimulated ATPase and had the in situ morphological characteristics of tonoplast including the presence of flocculent contents. These vesicles neither bound NPA nor contained levels of glucan synthetase II above background. Other membranous cell components such as dictyosomes (fucosyltransferase, latent nucleosidediphosphate phosphatase), endoplasmic reticulum vesicles (NADH- and NADPH- cytochrome c reductase), mitochondria (succinate-2(p-indophenyl)-3-p-nitrophenyl)-5-phenyl tetrazolium-reductase and cytochrome oxidase) and plastids (carotenoids and monogalactosyl diglyceride synthetase) were identified on the basis of appropriate marker constituents and, except for plastid thylakoids, had thin (<7 nanometers) membranes. They were located in the fractions intermediate between plasma membrane and tonoplast after free-flow electrophoretic separation and did not contaminate either the plasma membrane or the tonoplast fraction as determined from marker activities. From electron microscope morphometry (using both membrane measurements and staining with phosphotungstic acid at low pH) and analysis of marker enzymes, both plasma membrane and tonoplast fractions were estimated to be about 90% pure. Neither fraction appeared to be contaminated by the other by more than 3%.
本文描述了一种通过制备型自由流动电泳技术从黑暗生长的大豆（Glycine max L. var Wayne）下胚轴中分离出高纯度质膜和液泡膜组分的方法。向阳极迁移最慢的组分富含厚（10纳米）膜，基于其结合N-1-萘基邻苯二甲酸（NPA）、葡聚糖合成酶-II和K(+)-刺激、钒酸盐抑制的Mg(2+) ATP酶的能力、在电子显微镜切片上低pH下与磷钨酸的反应以及形态学评估，这些厚膜被鉴定为质膜。向阳极迁移最远（离样品注入点最远）的组分富含膜泡，其膜厚（7-9纳米），在低pH下不被磷钨酸染色，含有硝酸盐抑制、Cl-刺激的ATP酶，并且具有液泡膜的原位形态特征，包括存在絮状内含物。这些膜泡既不结合NPA，葡聚糖合成酶II的含量也不高于背景水平。其他膜性细胞成分，如高尔基体（岩藻糖基转移酶、潜在的核苷二磷酸磷酸酶）、内质网泡（NADH-和NADPH-细胞色素c还原酶）、线粒体（琥珀酸-2(p-吲哚苯基)-3-p-硝基苯基)-5-苯基四氮唑还原酶和细胞色素氧化酶）以及质体（类胡萝卜素和单半乳糖基甘油二酯合成酶），是根据适当的标记成分鉴定的，除了质体类囊体膜外，它们的膜都很薄（<7纳米）。在自由流动电泳分离后，它们位于质膜和液泡膜之间的组分中，根据标记活性测定，它们不会污染质膜或液泡膜组分。通过电子显微镜形态测量（使用膜测量和低pH下磷钨酸染色）以及标记酶分析，质膜和液泡膜组分的纯度估计均约为90%。两个组分似乎都未被另一个组分污染超过3%。
