Grassl S M
Department of Pharmacology, State University of New York Health Science Center, Syracuse 13210.
J Biol Chem. 1992 Sep 5;267(25):17760-5.
Membrane transport pathways for transplacental transfer of the water-soluble vitamin biotin were investigated by assessing the possible presence of a Na(+)-biotin cotransport mechanism in the maternal-facing membrane of human placental epithelial cells. The presence of Na(+)-biotin cotransport was determined from radiolabeled tracer flux measurements of biotin uptake using preparations of purified brush-border membrane vesicles. The imposition of an inwardly directed Na+ gradient stimulated vesicle uptake of biotin to levels approximately 25-fold greater than those observed at equilibrium. The voltage sensitivity of Na+ gradient-driven biotin uptake suggested Na(+)-biotin cotransport is electrogenic occurring with net transfer of positive charge. A kinetic analysis of the activation of biotin uptake by increasing Na+ was most consistent with an interaction of Na+ at 2 sites in the transport protein. Static head determinations used to identify the magnitude of opposing driving forces bringing flux through the cotransport mechanism to equilibrium indicated a coupling ratio of 2 Na+ per biotin. Substrate specificity studies using chemical analogues of biotin suggested both the terminal carboxylic acid of the valeric acid side chain and a second nucleus of anionic charge were important determinants for substrate interaction with the cotransport protein. Initial rate determinations of biotin uptake indicate biotin interacts with a single saturable site (Km = 21 microM) with a maximal transport rate of 4.5 nmol/mg/min. The results of this study provide evidence for an electrogenic Na(+)-biotin cotransport mechanism in the maternal-facing membrane of human placental epithelial cells.
通过评估人胎盘上皮细胞母体侧膜中可能存在的Na⁺-生物素共转运机制，研究了水溶性维生素生物素经胎盘转运的膜转运途径。使用纯化的刷状缘膜囊泡制剂，通过生物素摄取的放射性标记示踪剂通量测量来确定Na⁺-生物素共转运的存在。内向的Na⁺梯度的施加刺激了生物素囊泡摄取，使其水平比在平衡时观察到的水平高约25倍。Na⁺梯度驱动的生物素摄取的电压敏感性表明Na⁺-生物素共转运是生电的，伴随着正电荷的净转移。通过增加Na⁺对生物素摄取激活的动力学分析最符合Na⁺在转运蛋白中的2个位点相互作用。用于确定使通过共转运机制的通量达到平衡的相反驱动力大小的静态头测定表明，每生物素的耦合比为2个Na⁺。使用生物素化学类似物的底物特异性研究表明，戊酸侧链的末端羧酸和第二个阴离子电荷核都是底物与共转运蛋白相互作用的重要决定因素。生物素摄取的初始速率测定表明，生物素与单个可饱和位点（Km = 21 μM）相互作用，最大转运速率为4.5 nmol/mg/min。本研究结果为人类胎盘上皮细胞母体侧膜中存在生电的Na⁺-生物素共转运机制提供了证据。
