Monneron A, d'Alayer J
J Cell Biol. 1978 Apr;77(1):211-31. doi: 10.1083/jcb.77.1.211.
The purpose of this work was to isolate thymocyte plasma membranes at high yield and purity to study specific surface molecules in their structural context. A procedure was developed in which 92-95% of the cells were disrupted by homogenization in a dense viscous medium, while nuclei remained intact. Differential centrifugation of the homogenate was avoided; instead, only a brief (2 h) centrifugation at equilibrium-density of membrane components was used. Five fractions were obtained, three by flotation. Membrane-bound enzymatic activities indicated a 60-80% yield of plasma membranes in the three floated membrane fractions, which comprised 1.6% of the homogenate protein. Enrichment factors for three ectoenzymes, alkaline phosphatase, gamma-glutamyltransferase, and ouabain-sensitive adenosine triphosphatase were respectively, 70-74, and 40-50 in the two lightest fractions. Nuclear membranes were then isolated from the remaining whole nuclei and were found to be enriched in esterase and NADH-cytochrome c reductase. Plasma membranes and light nuclear membranes appeared as pure unit-membrane vesicles in thin sections and freeze-etching electron microscopy. Some aggregation of intramembranous particles occurred in plasma membrane vesicles.
这项工作的目的是高产率、高纯度地分离胸腺细胞质膜，以便在其结构背景下研究特定的表面分子。开发了一种方法，其中92% - 95%的细胞在浓稠的粘性介质中通过匀浆被破坏，而细胞核保持完整。避免了对匀浆进行差速离心；相反，仅在膜成分的平衡密度下进行短暂（2小时）的离心。获得了五个组分，其中三个是通过浮选得到的。膜结合酶活性表明，在三个浮选的膜组分中质膜的产率为60% - 80%，这些组分占匀浆蛋白的1.6%。两种最轻组分中三种外切酶（碱性磷酸酶、γ - 谷氨酰转移酶和哇巴因敏感的三磷酸腺苷酶）的富集系数分别为70 - 74和40 - 50。然后从剩余的完整细胞核中分离出核膜，发现其富含酯酶和NADH - 细胞色素c还原酶。在超薄切片和冷冻蚀刻电子显微镜下，质膜和轻核膜呈现为纯净的单位膜囊泡。质膜囊泡中发生了一些膜内颗粒的聚集。
