Proteomic evaluation of plasma membrane fraction prepared from mouse liver and kidney using a bead homogenizer: Enrichment of drug-related transporter proteins.,Molecular Pharmaceutics

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Proteomic evaluation of plasma membrane fraction prepared from mouse liver and kidney using a bead homogenizer: Enrichment of drug-related transporter proteins.
Molecular Pharmaceutics ( IF 4.5 ) Pub Date : 2020-09-09 , DOI: 10.1021/acs.molpharmaceut.0c00547
Ryotaro Yagi ₁ , Takeshi Masuda _{1,

2,

3} , Seiryo Ogata ₁ , Ayano Mori ₃ , Shingo Ito _{1,

2,

3} , Sumio Ohtsuki _{1,

2,

3}

Affiliation

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 Na^+/K⁺-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.

中文翻译：

使用珠子匀浆器从小鼠肝脏和肾脏制备的质膜组分的蛋白质组学评估：药物相关转运蛋白的富集。

量化质膜组分中药物转运蛋白的蛋白质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子匀浆器对从小鼠肝脏和肾脏组织制备的质膜组分中富集的药物相关转运蛋白进行了蛋白质组学评估。使用 Dounce 或珠子匀浆器制备粗制和质膜级分，并使用定量蛋白质组学确定蛋白质水平。在肝组织中，质膜组分比粗膜组分更富含转运蛋白；使用 Dounce 和珠子均质器，血浆与粗膜组分的平均富集比分别为 3.31 和 6.93。使用珠子匀浆器测定的质膜组分中转运蛋白的浓度高于使用 Dounce 匀浆器测定的浓度。同时，在肾组织中，两种匀浆器的质膜组分都富含位于刷状缘膜中的转运蛋白。然而，使用任一均质器获得的膜级分均不富含 Na^+/ K ⁺ -ATPase 和转运蛋白定位于基底外侧膜。这些结果表明，使用珠匀浆器进行分级分离，可以从小鼠肝脏和肾脏组织中产生富含转运蛋白的质膜组分；然而，在从肾组织制备的质膜组分中没有观察到基底外侧转运蛋白的富集。

更新日期：2020-11-02

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