Department of Pharmaceutical Microbiology, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan.
Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan.
Mol Pharm. 2020 Nov 2;17(11):4101-4113. doi: 10.1021/acs.molpharmaceut.0c00547. Epub 2020 Oct 2.
Quantifying the protein levels of drug transporters in plasma membrane fraction helps elucidate the function of these transporters. In this study, we conducted a proteomic evaluation of enriched drug-related transporter proteins in plasma membrane fraction prepared from mouse liver and kidney tissues using the membrane protein extraction kit and a bead homogenizer. Crude and plasma membrane fractions were prepared using either the Dounce or bead homogenizer, and protein levels were determined using quantitative proteomics. In liver tissues, the plasma membrane fractions were more enriched in transporter proteins than the crude membrane fractions; the average enrichment ratios of plasma-to-crude membrane fractions were 3.31 and 6.93 using the Dounce and bead homogenizers, respectively. The concentrations of transporter proteins in plasma membrane fractions determined using the bead homogenizer were higher than those determined using the Dounce homogenizer. Meanwhile, in kidney tissues, the plasma membrane fractions were enriched in transporters localized in the brush-border membrane to the same degree for both the homogenizers; however, the membrane fractions obtained using either homogenizer were not enriched in NaK-ATPase and transporters localized in the basolateral membrane. These results indicate that fractionation, using the bead homogenizer, yielded transporter-enriched plasma membrane fractions from mouse liver and kidney tissues; however, no enrichment of basolateral transporters was observed in plasma membrane fractions prepared from kidney tissues.
定量分析质膜部分中药物转运蛋白的蛋白水平有助于阐明这些转运蛋白的功能。在这项研究中，我们使用膜蛋白提取试剂盒和珠匀浆机，对从小鼠肝和肾组织中制备的富含药物相关转运蛋白的质膜部分进行了蛋白质组学评估。使用 Dounce 匀浆器或珠匀浆器分别制备粗质膜部分和质膜部分，并使用定量蛋白质组学方法测定蛋白水平。在肝组织中，质膜部分比粗质膜部分更富含转运蛋白；使用 Dounce 匀浆器和珠匀浆器，质膜部分相对于粗质膜部分的平均富集比分别为 3.31 和 6.93。使用珠匀浆器测定的质膜部分转运蛋白浓度高于使用 Dounce 匀浆器测定的浓度。同时，在肾组织中，两种匀浆器都使刷状缘膜定位的转运蛋白在质膜部分得到相同程度的富集；然而，两种匀浆器获得的膜部分都没有使 NaK-ATP 酶和基底外侧膜定位的转运蛋白得到富集。这些结果表明，使用珠匀浆器进行的分级分离从小鼠肝和肾组织中产生了富含转运蛋白的质膜部分；然而，在从肾组织制备的质膜部分中未观察到基底外侧转运蛋白的富集。
