Bottà G, Meyer G, Rossetti C, Cremaschi D
Biochim Biophys Acta. 1987 Feb 26;897(2):315-23. doi: 10.1016/0005-2736(87)90427-5.
The apical membranes of rabbit gallbladder epithelial cells were isolated by treating the homogenate with Ca2+ or Mg2+ and centrifuging the suspension in Percoll gradient. In this way brush-border membranes were obtained with enrichment factors ranging between 10 and 20 and yields of 15-30%. A second method is described with which membranes were isolated, without any preliminary treatment, first by differential centrifugation, then with Percoll gradient; the final membrane enrichment was over 15, however the yield was very low (3%). Many possible enzymatic markers of the apical plasma membrane were investigated: L-gamma-glutamyltransferase, alkaline phosphatase, leucine aminopeptidase, sucrase. The first appears to be that of choice. Apical membrane fraction could be also evidenced by autofluorescence or by labeling with Lotus tetragonolobus lectin. Preliminary experiments showed that apical plasma membranes isolated in this way form vesicles.
通过用Ca2+或Mg2+处理匀浆并在Percoll梯度中离心悬浮液，分离出兔胆囊上皮细胞的顶端膜。通过这种方法获得的刷状缘膜的富集因子在10至20之间，产率为15 - 30%。描述了另一种方法，即不经任何预处理，首先通过差速离心，然后通过Percoll梯度分离膜；最终膜富集超过15，但产率非常低(3%)。研究了许多可能的顶端质膜酶标记物：L-γ-谷氨酰转移酶、碱性磷酸酶、亮氨酸氨肽酶、蔗糖酶。第一种似乎是首选。顶端膜部分也可以通过自发荧光或用百脉根凝集素标记来证明。初步实验表明，以这种方式分离的顶端质膜形成囊泡。
