Brasitus T A, Keresztes R S
Biochim Biophys Acta. 1984 Jun 27;773(2):290-300. doi: 10.1016/0005-2736(84)90093-2.
The isolation of apical membranes from rat proximal colonic epithelial cells is described. Differential centrifugation yielded a 'crude' membrane fraction which was further purified using sucrose density centrifugation. The final membrane fraction was enriched 20-28-fold over homogenate in alkaline phosphatase and cysteine-sensitive alkaline phosphatase specific activities. Lipid-protein interactions and lipid dynamics examined in apical and basolateral membranes prepared from colonocytes demonstrated: (1) apical membrane, as assessed by steady-state fluorescence polarization studies have a low lipid fluidity; (2) colonic basolateral membranes possess a greater lipid fluidity than apical membranes; (3) compositional differences in these antipodal membranes appear to explain these differences in lipid fluidity; (4) fluorescence polarization studies using diphenylhexatriene detect a thermotropic transition at 21-23 degrees C in apical membranes and liposomes prepared from lipid extracts of these membranes; (5) alkaline phosphatase and L-cysteine-sensitive alkaline phosphatase activities appear to be functionally dependent on the physical state of the apical membrane's lipid.
本文描述了从大鼠近端结肠上皮细胞中分离顶端膜的方法。差速离心得到一个“粗”膜组分，该组分再通过蔗糖密度离心进一步纯化。最终的膜组分在碱性磷酸酶和半胱氨酸敏感碱性磷酸酶的比活性方面比匀浆富集了20 - 28倍。对结肠细胞制备的顶端膜和基底外侧膜中的脂 - 蛋白相互作用及脂质动力学进行研究，结果表明：（1）通过稳态荧光偏振研究评估，顶端膜的脂质流动性较低；（2）结肠基底外侧膜的脂质流动性高于顶端膜；（3）这些对向膜的组成差异似乎解释了脂质流动性的这些差异；（4）使用二苯基己三烯的荧光偏振研究在顶端膜以及由这些膜的脂质提取物制备的脂质体中检测到在21 - 23℃有一个热致转变；（5）碱性磷酸酶和L - 半胱氨酸敏感碱性磷酸酶的活性似乎在功能上依赖于顶端膜脂质的物理状态。
