Dyer D L, Said H M
VA Medical Center, Research Service, Long Beach, CA 90822.
Biochim Biophys Acta. 1995 Mar 8;1234(1):15-21. doi: 10.1016/0005-2736(94)00232-e.
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.
在非洲爪蟾卵母细胞中证实了存在一种与膜相关的核黄素（RF）摄取载体。低浓度（0.017微摩尔）和高浓度（3微摩尔）RF的摄取在长达2小时内与时间呈线性关系，且最初与卵母细胞的结合很少，代谢也很少。发现RF的摄取与细胞外pH值和Na+无关。RF摄取的初始速率作为浓度的函数是可饱和的，表观Km为0.41±0.02微摩尔，Vmax为2.86±0.04飞摩尔/卵母细胞每小时。3H-RF的摄取受到未标记的RF以及结构类似物发光黄素、异核黄素（异-RF）、8-氨基核黄素（8-NH2-RF）、8-羟基核黄素（8-OH-RF）和色素黄的抑制，但不受黄素腺嘌呤二核苷酸（FAD）、D-核糖或鲁米嗪的影响。RF的摄取受到代谢抑制剂2,4-二硝基苯酚的显著抑制。巯基修饰试剂对氯汞苯磺酸盐（pCMPS）、对氯汞苯甲酸（pCMB）、N-乙基马来酰亚胺和7-氯-4-硝基苯并-2-恶唑-1,3-二氮杂环戊二烯（NBD-Cl）均对RF摄取有显著抑制作用。用还原剂处理经pCMPS预处理的细胞可完全逆转pCMPS的抑制作用。虽然跨膜转运抑制剂4-乙酰氨基-4'-异硫氰酸基芪-2,2'-二磺酸（SITS）、4,4'-二异硫氰酸基芪-2,2'-二磺酸（DIDS）和速尿对RF摄取无影响，但氨氯地平和丙磺舒以剂量依赖方式抑制RF摄取。对氨氯地平介导的抑制作用的进一步研究表明，其本质上是竞争性的，表观Ki约为1.8毫摩尔，而丙磺舒诱导的抑制是非特异性的。总之，这些发现表明非洲爪蟾卵母细胞拥有一种内源性、特异性、与膜相关的载体介导的RF摄取系统。结果还证明了非洲爪蟾卵母细胞作为研究RF跨生物膜转运事件的模型系统的有用性，这应进一步加深我们对各种脊椎动物细胞摄取RF的理解。
