Proteomic Evaluation of Plasma Membrane Fraction Prepared from a Mouse Liver and Kidney Using a Bead Homogenizer: Enrichment of Drug-Related Transporter Proteins.

Abstract

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.