Biochimica et Biophysica Acta vol:979 issue:1 pages:91-98
The characteristics of Na+-dependent hexose uptake were determined for monolayers of OK, an established renal epithelial cell line derived from an opossum kidney. A comparison is made with other cultured cells, particularly LLC-PK1. The capacity to accumulate alpha-methyl D-glucoside (AMG) in OK cells develops with time, reaching a maximum level of 18 nmol/mg protein per h, 3 days after confluency. In contrast to LLC-PK1, this level is not influenced by the medium D-glucose concentration. AMG uptake in OK cells was characterized by an apparent Km of 2.9 mM and a Vmax of 17.1 nmol/mg protein per min. For Na+-dependent phlorisin binding, a KD of 0.025 microM and a Bmax of 1.5 pmol/mg protein were found. A turnover frequency of 158/s was derived from our data. The hexose carrier of OK shares with the carrier of LLC-PK1 a high level of expression, its substrate specificity and turnover frequency. It differs however with respect to the substrate binding site. The affinity for AMG and D-glucose is 3- and 10-fold lower, whereas the affinity for phlorizin is 3-times higher in OK than in LLC-PK1. The Na+ dependence of AMG uptake was also different for both cell lines and suggested for OK cells a 1:1, Na+:substrate stoichiometry. In OK cells, the phlorizin-sensitive uptake rate of D-glucose is much lower than the one for AMG. Nevertheless, D-glucose interacts with the AMG binding site in a competitive way and with an affinity similar to AMG. This could indicate a malfunction of the carrier with D-glucose as a substrate at the level of the translocation step.