TAILIEUCHUNG - Báo cáo khoa học: Transport of the phosphonodipeptide alafosfalin by the H+/peptide cotransporters PEPT1 and PEPT2 in intestinal and renal epithelial cells

The interaction of the antibacterial phosphonodipeptide alafosfalinwithmammalianH + /peptide cotransporters was studied in Caco-2 cells, expressing the low-affinity intestinal type peptide transporter 1 (PEPT1), and SKPT cells, expressing the high-affinity renal type peptide transporter 2 (PEPT2). Alafosfalin strongly inhibited the uptake of [ 14 C]glycylsarcosine withKi values of ± mMand ± mMfor PEPT1 and PEPT2, respectively. | Eur. J. Biochem. 271 2012-2017 2004 FEBS 2004 doi Transport of the phosphonodipeptide alafosfalin by the H peptide cotransporters PEPT1 and PEPT2 in intestinal and renal epithelial cells Jana Neumann1 Mandy Bruch1 Sabine Gebauer2 and Matthias Brandsch1 1Membrane Transport Group Biozentrum and 2Institute of Biochemistry Department of BiochemistryỊBiotechnology Martin-Luther-University Halle-Wittenberg Halle Germany The interaction of the antibacterial phosphonodipeptide alafosfalin with mammalian H peptide cotransporters was studied in Caco-2 cells expressing the low-affinity intestinal type peptide transporter 1 PEPT1 and SKPT cells expressing the high-affinity renal type peptide transporter 2 PEPT2 . Alafosfalin strongly inhibited the uptake of 14C glycylsarcosine with Ki values of mM and mM for PEPT1 and PEPT2 respectively. Saturation kinetic studies revealed that in both cell types ala-fosfalin affected only the affinity constant Kt but not the maximal velocity Vmax of glycylsarcosine Gly-Sar uptake. The inhibition constants and the competitive nature of inhibition were confirmed in Dixon-type experiments. Caco-2 cells and SKPT cells were also cultured on permeable filters apical uptake and transepithelial apical to basolateral flux of 14C Gly-Sar across Caco-2 cell monolayers were reduced by alafosfalin 3 mM by 73 . In SKPT cells uptake of 14C Gly-Sar but not flux was inhibited by 61 . We found no evidence for an inhibition of the basolateral to apical uptake or flux of 14C Gly-Sarby alafosfalin. Alafosfalin 3 mM did not affect the apical to basolateral 14C mannitol flux. Determined in an Ussing-type experiment with Caco-2 cells cultured in SnapwellsTM alafosfalin increased the shortcircuit current through Caco-2 cell monolayers. We conclude that alafosfalin interacts with both H peptide symporters and that alafosfalin is actively transported across the intestinal epithelium in a H -symport explaining its .

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