RIBOFLAVIN TRANSPORT BY RABBIT RENAL BRUSH-BORDER MEMBRANE-VESICLES

The present study examined riboflavin (RF) uptake by isolated rabbit r enal brush border membrane (BBM), RF uptake was linear for up to 30 s and leveled off thereafter reaching an equilibrium with longer incubat ion. Studies on RF uptake as a function of incubation medium osmolarit y indicated that the uptake was the results of transport (61.4%) into the intravesicular space as well as binding (38.6%) to membrane surfac es. The process of RF uptake was saturable as a function of substrate concentration with an apparent K-m of 25.7 +/- 7.6 mu M and V-max of 7 5.6 +/- 14.7 pmol/mg protein/10 s. cis-Addition of unlabeled RF and it s structural analogues, lumiflavin and lumichrome, inhibited the uptak e of [H-3]RF significantly, indicating the involvement of a carrier-me diated process in RF uptake by renal BBM. RF uptake by renal BBM was p artly Na+-dependent so that when Na+ was replaced by potassium, cholin e, lithium or tetramethylammonium, the RF uptake was reduced to ca. 60 % of the control. This Na+-dependency was unlikely to be due to Na+-co transport mechanism because RF uptake occurred without the characteris tic 'overshoot' phenomenon as for other Na+-cotransport systems and th e elimination of transmembrane Na+-gradient by preloading Na+ to the i ntravesieular space did not affect RF uptake. In contrast, removal of Na+ eliminated the binding component of RF uptake, suggesting the requ irement of Na+ for RF binding to BBM. The RF uptake was not affected w hen extravesicular pH was varied within the physiological pH range of 6.5 to 8.5. No effect on BBM [H-3]RF uptake was found when the transme mbrane electrical potential was altered by either the presence of anio ns with different membrane permeability (Cl- = NO3- > SO4- > gluconate (-)) or by using nigericin (10 mu g/mg protein) with an outwardly or i nwardly directed transmembrane K+ gradient, The uptake of RF by BBM ve sicles was, however, inhibited by probenecid and organic anion transpo rt inhibitors, 4,4-diiso-thiscyanatostilbene-2,2-disulfonic acid (DIDS , 1 mM) and -acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid(SI TS, 1 mM), In summary, these results demonstrate the existence of a me mbrane-associated, and organic anion inhibitor-sensitive, carrier syst em for RF uptake by renal BBM. (C) 1997 Elsevier Science B.V.