Riboflavin uptake by native Xenopus laevis oocytes.
The existence of a membrane-associated uptake carrier for riboflavin (RF) is demonstrated in Xenopus oocytes. Uptake of low (0.017 microM) and high (3 microM) concentrations of RF was linear with time for up to 2 hours, and occurred with little initial binding to oocytes, and little metabolism. Uptake of RF was found to be independent of extracellular pH and Na+. The initial rate of RF uptake was saturable as a function of concentration with an apparent Km of 0.41 +/- 0.02 microM and a Vmax of 2.86 +/- 0.04 fmol/oocyte per h. Uptake of 3H-RF was inhibited by unlabeled RF and by the structural analogs lumiflavin, isoriboflavin (iso-RF), 8-aminoriboflavin (8-NH2-RF), 8-hydroxyriboflavin (8-OH-RF), and lumichrome, but was not affected by flavin adenine dinucleotide (FAD), D-ribose or lumazine. Uptake of RF was significantly retarded by the metabolic inhibitor 2,4-dinitrophenol. The sulfhydryl group-modifying reagents p-chloromercuriphenylsulfonate (pCMPS), p-chloromercuribenzoate (pCMB), N-ethylmaleimide and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) all caused significant inhibition in RF uptake. The inhibitory effect of pCMPS was completely reversed by treatment of pCMPS-pretreated cells with reducing agents. While the transmembrane transport inhibitors 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and furosemide had no effect on RF uptake, amiloride and probenecid suppressed RF uptake in a dose-dependent fashion. Closer examination of the inhibition mediated by amiloride showed that it was competitive in nature with an apparent Ki of approximately 1.8 mM, whereas the inhibition induced by probenecid was nonspecific. Together, these findings indicate that Xenopus oocytes possess an endogenous, specific, membrane-associated carrier-mediated uptake system for RF. The results also demonstrate the usefulness of Xenopus oocytes as a model system with which to study the RF transport event across biological membranes, which should further out present understanding of RF uptake by various vertebrate cells.
Dyer
D L
DL
VA Medical Center, Research Service, Long Beach, CA 90822.
Said
H M
HM
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D001693:Biological Transport, Active / Q000187:drug effects; D066298:In Vitro Techniques; D007700:Kinetics; D009865:Oocytes / Q000378:metabolism*; D012256:Riboflavin / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014982:Xenopus laevis
D013439:Sulfhydryl Reagents; D000584:Amiloride; D012256:Riboflavin