Abstract
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.
Highlights
Membrane transportpathwaysfortransplacental the elucidation of transport pathways located at thebasal or transfer of the water-soluble vitamin biotin were in- fetal-facing membrane may be expedited by recent devestigated by assessing the possible presence of a Na+biotin cotransport mechanism in the maternal-facing membrane of humanplacentalepithelial cells
This study provide evidence for an electrogenic Na+- Membrane Preparations-Brush-border membrane vesicles were biotin cotransport mechanism in the maternal-facing isolated from human term placenta by divalent cation aggregation membrane of human placental epitheliacl ells
Nu' Gradient-driven Biotin Influx-The possible presence tion gradient and thienflux of biotin was investigated further of a Na'-biotin cotransport mechanism in human placental by observing theeffect of gramicidin on Na' gradient-driven brush-border membrane was first investigated by assessing biotin uptake.To the extent thNaat' gradient-induced biotin theability of a Na' concentrationgradienttoserveas a uptake occurred via a transmembrane conductive pathway for driving force for intravesicular concentrative accumulationof biotin coupled to aninside-positive Na' diffusion potential, a biotin
Summary
Membrane transportpathwaysfortransplacental the elucidation of transport pathways located at thebasal or transfer of the water-soluble vitamin biotin were in- fetal-facing membrane may be expedited by recent devestigated by assessing the possible presence of a Na+biotin cotransport mechanism in the maternal-facing membrane of humanplacentalepithelial cells. Nu' Gradient-driven Biotin Influx-The possible presence tion gradient and thienflux of biotin was investigated further of a Na'-biotin cotransport mechanism in human placental by observing theeffect of gramicidin on Na' gradient-driven brush-border membrane was first investigated by assessing biotin uptake.To the extent thNaat' gradient-induced biotin theability of a Na' concentrationgradienttoserveas a uptake occurred via a transmembrane conductive pathway for driving force for intravesicular concentrative accumulationof biotin coupled to aninside-positive Na' diffusion potential, a biotin.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have