Brown C D, Bodmer M, Biber J, Murer H
Biochim Biophys Acta. 1984 Jan 25;769(2):471-8. doi: 10.1016/0005-2736(84)90332-8.
Apical membrane vesicles were prepared from confluent monolayers of LLC-PK1 cells grown upon microcarrier beads. The final membrane preparation, obtained by a modified divalent cation precipitation technique, was enriched in alkaline phosphatase, leucine aminopeptidase and trehalase (8-fold compared to the initial homogenate). Analysis of phosphate uptake into the vesicles identified a specific sodium-dependent pathway. Lithium and other cations were unable to replace sodium. At 100 mmol/l sodium and pH 7.4, an apparent Km for phosphate of 99 +/- 19 mumol/l and an apparent Ki for arsenate of 1.9 mmol/l were found. Analysis of the sodium activation of phosphate uptake gave an apparent Km for sodium of 32 +/- 12 mmol/l and suggested the involvement of two sodium ions in the transport mechanism. Sodium modified the apparent Km of the transport system for phosphate. The rate of sodium-dependent phosphate uptake was higher at pH 6.4 than at pH 7.4. At both pH values, an inside negative membrane potential (potassium gradient plus valinomycin) had no stimulatory effect on the rate of the sodium-dependent component of phosphate uptake. It is concluded that the apical membrane of LLC-PK1 cells contains a sodium-phosphate cotransport system with a stoichiometry of 2 sodium ions: 1 phosphate anion.
从生长在微载体珠上的LLC - PK1细胞汇合单层制备顶端膜囊泡。通过改良的二价阳离子沉淀技术获得的最终膜制剂，碱性磷酸酶、亮氨酸氨肽酶和海藻糖酶含量丰富（与初始匀浆相比增加了8倍）。对囊泡中磷酸盐摄取的分析确定了一种特定的钠依赖性途径。锂和其他阳离子不能替代钠。在100 mmol/L钠和pH 7.4条件下，发现磷酸盐的表观Km为99±19 μmol/L，砷酸盐的表观Ki为1.9 mmol/L。对磷酸盐摄取的钠激活分析得出钠的表观Km为32±12 mmol/L，并表明转运机制中有两个钠离子参与。钠改变了磷酸盐转运系统的表观Km。在pH 6.4时，钠依赖性磷酸盐摄取速率高于pH 7.4时。在两个pH值下，内向负膜电位（钾梯度加缬氨霉素）对磷酸盐摄取的钠依赖性成分的速率均无刺激作用。结论是，LLC - PK1细胞的顶端膜含有一种钠 - 磷酸盐共转运系统，化学计量比为2个钠离子：1个磷酸根阴离子。
