【原著】 膜小胞および培養細胞を用いた生体膜輸送研究のための新規反応停止液の開発

Abstract

There are certain transporters that function effectively even at ice–cold temperatures, such as facilitative glucose transporter (GLUT) 1 and equilibrative nucleoside transporter (ENT) 1 in human erythrocyte membranes. In order to evaluate the characteristics of these transporters precisely, inhibition of substrate efflux during stopping and washing processes is essential. In this study, we attempted to develop a novel stop solution that can be used universally for membrane transport studies. As candidate compounds for preparing a novel stop solution, protein denaturants such as urea, 2–mercaptoethanol, formaldehyde, and glutaraldehyde were used. D–Glucose and uridine were used as substrates for GLUT1 and ENT1, respectively. [3H]D–Glucose taken up by rightside–out erythrocyte membrane vesicles (ROVs) was almost completely effluxed during washing with ice–cold stop solution without phloretin. Protein denaturants inhibited the efflux of these substrates, and increased the remaining amount of substrates in ROVs. Especially, the combination of urea/formaldehyde or 2–mercaptoethanol/glutaraldehyde was effective as a stop solution for both GLUT1– and ENT1–mediated efflux. In addition, these stop solutions were applicable for the transport study in cultured HepG2 cells. Our results indicate that these novel stop solutions can be used universally for membrane transport studies with isolated membrane vesicles and culture cells.