Isolation and characterization of the Xenopus oocyte plasma membrane:: a new method for studying activity of water and solute transporters
Isolation and characterization of the Xenopus oocyte plasma membrane:: a new method for studying activity of water and solute transporters
复制标题
DOI：
10.1152/ajprenal.00022.2005
复制
发表时间：
2005-07-01
影响因子：
4.2
通讯作者：
Zeidel, ML
中科院分区：
文献类型：
--
作者：
Hill, WG;Southern, NM;Zeidel, ML
非洲爪哇完整的卵母细胞是研究表达的水和溶质转运蛋白的有用的模型系统，但受到一些限制，最明显的是大的未搅拌的层和其他细胞内的扩散障碍。为了克服这些问题，我们开发了一种从卵母细胞中分离质膜囊泡的方法。这种方法有助于更精确地控制囊泡内环境，并几乎消除了动力学实验中未搅拌层的问题。与匀浆相比，分离过程使质膜标记碱性磷酸二酯酶的丰度增加了50.6倍。晚期内质体/溶酶体和线粒体的标志物没有丰富，内质网仅轻度丰富。天然质膜对水和尿素的透过率分别为8.1x10(-4)和5.6x10(-7)cm/S，足以将其归类为屏障膜。质谱仪分析表明，不含胆固醇的膜富含磷脂酰胆碱(35.8%)、鞘磷脂(25.8%)和磷脂酰肌醇(6.8%)。胆固醇浓度为20.7%。在停流实验中，从表达水通道蛋白-1的卵母细胞中分离出的膜小泡的透水性增加了四倍。表达小鼠尿素转运蛋白A3(UT-A3)的卵母细胞表现出比注水对照组快7.5倍的速度。UT-A3和UT-A3的膜透性无明显差异，提示UT-A3不是水载体。总之，我们描述了一种分离卵母细胞质膜的改进方法，该方法将允许研究水和溶质运输动力学以及异源表达蛋白质的底物选择性。
The intact Xenopus laevis oocyte is a useful model system for studying expressed water and solute transporters but suffers from a number of limitations, most notably large unstirred layers and other intracellular diffusion barriers. To overcome these, we have developed a method for isolating plasma membrane vesicles from oocytes. This approach facilitates more precise control of the intravesicular environment and virtually eliminates the problem of unstirred layers in kinetic experiments. The isolation procedure results in 50.6-fold enrichment of the plasma membrane marker alkaline phosphodiesterase compared with the homogenate. Markers of late endosomes/lysosomes and mitochondria were not enriched, and the endoplasmic reticulum was enriched only modestly. Permeabilities of native plasma membrane to water and urea were 8.1 x 10(-4) and 5.6 x 10(-7) cm/s, respectively, values that are sufficiently low to classify them as barrier membranes. Phospholipid analysis by mass spectrometry showed the membrane, not including cholesterol, to be rich in phosphatidylcholine (35.8 mole percent), sphingomyelin (25.8 mole percent), and phosphatidylinositol (6.8 mole percent). Cholesterol concentration was 20.7 mole percent. Membrane vesicles isolated from oocytes expressing aquaporin-1 exhibited fourfold higher water permeability in stopped-flow experiments. Oocytes expressing mouse urea transporter A3 (UT-A3) exhibited 7.5-fold faster phloretin-inhibitable urea transport compared with water-injected controls. There was no difference in water permeability between these membrane vesicles, suggesting that UT-A3 is not a water carrier. In conclusion, we describe an improved method for the isolation of the oocyte plasma membrane that will allow the study of water and solute transport kinetics as well as substrate selectivity in heterologously expressed proteins.