Musílková J, Kriegerbecková K, Krůsek J, Kovár J
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Biochim Biophys Acta. 1998 Feb 2;1369(1):103-8. doi: 10.1016/s0005-2736(97)00214-9.
We studied transport of non-transferrin iron into HeLa cells adapted for growth in defined medium, containing either 5 micrograms/ml of iron-saturated transferrin (HeLa/Tf cells) or 5 microM ferric citrate (HeLa/Fe5 cells) as a source of iron. Employing 55Fe-ferric citrate, iron uptake by intact cells was compared with iron binding to isolated membranes. Uptake characteristics of both HeLa/Tf and HeLa/Fe5 cells seemed to be similar: Km = 14 microM and Vmax = 135 pmol Fe/min/10(5) cells for HeLa/Tf, Km = 22 microM and Vmax = 165 pmol Fe/min/10(5) cells for HeLa/Fe5. Increasing concentrations (0.3-1.2 microM) of 55Fe-ferric citrate, producing levels of free 55Fe which were independent of total Fe under the experimental conditions used, led to increased binding of 55Fe for both HeLa/Tf and HeLa/Fe5 cells (1.08-8.03 nmol Fe/h/10(5) cells). This corresponds with the suggestion that iron was bound in the form of ferric citrate rather than in the form of free iron. Dissociation constants of Fe binding, KD = 0.61 microM for HeLa/Tf and KD = 1.53 microM for HeLa/Fe5, were obtained from competition experiments. We conclude that specific binding sites for ferric citrate are constitutively expressed in plasma membrane and that their expression does not require the induction by the presence of ferric citrate. The uptake of non-transferrin iron is realized in at least two steps. The first step is iron binding to the specific binding sites in plasma membrane. The binding does not represent a limiting step of the uptake.
我们研究了非转铁蛋白铁进入适应在限定培养基中生长的HeLa细胞的转运情况，该培养基含有5微克/毫升的铁饱和转铁蛋白（HeLa/Tf细胞）或5微摩尔柠檬酸铁（HeLa/Fe5细胞）作为铁源。使用55Fe - 柠檬酸铁，将完整细胞的铁摄取与铁与分离膜的结合进行了比较。HeLa/Tf和HeLa/Fe5细胞的摄取特性似乎相似：HeLa/Tf细胞的Km = 14微摩尔，Vmax = 135皮摩尔铁/分钟/10^5个细胞；HeLa/Fe5细胞的Km = 22微摩尔，Vmax = 165皮摩尔铁/分钟/10^5个细胞。在所用实验条件下，增加55Fe - 柠檬酸铁的浓度（0.3 - 1.2微摩尔），产生与总铁无关的游离55Fe水平，导致HeLa/Tf和HeLa/Fe5细胞对55Fe的结合增加（1.08 - 8.03纳摩尔铁/小时/10^5个细胞）。这与铁以柠檬酸铁形式而非游离铁形式结合的观点相符。通过竞争实验获得了Fe结合的解离常数，HeLa/Tf细胞的KD = 0.61微摩尔，HeLa/Fe5细胞的KD = 1.53微摩尔。我们得出结论，柠檬酸铁的特异性结合位点在质膜中组成性表达，其表达不需要柠檬酸铁的诱导。非转铁蛋白铁的摄取至少通过两个步骤实现。第一步是铁与质膜中的特异性结合位点结合。该结合并不代表摄取的限速步骤。
