Oshiro S, Nakajima H, Markello T, Krasnewich D, Bernardini I, Gahl W A
Section on Human Biochemical Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1993 Oct 15;268(29):21586-91.
Sepharose beads bound to 55Fe-transferrin (Tf) were used to evaluate Tf-dependent iron uptake not employing receptor-mediated endocytosis (RME). The iron of 55Fe2-Tf-Sepharose was reduced and taken up by cultured human fibroblasts in a time- and concentration-dependent fashion (Km 7 microM; Vmax 128 pmol/mg/min). This redox system resembled that for Tf-independent iron uptake (Tf-IU, evaluated using 55Fe-citrate) in several ways. 1) NH4Cl did not inhibit iron uptake from 55Fe-citrate and 55Fe2-Tf-Sepharose but did inhibit uptake from 55Fe2-Tf (RME system). 2) Iron uptake and reduction from 55Fe2-Tf-Sepharose and 55Fe-citrate increased with temperature hyperbolically, differing from the sigmoidal curve for RME uptake. 3) The subcellular distributions of iron from 55Fe-citrate and 55Fe2-Tf-Sepharose resembled each other and differed from that for 55Fe2-Tf. 4) The optimal pH for iron reduction and uptake using 55Fe2-Tf-Sepharose or 55Fe-citrate was less than pH 5.5, while that for iron uptake from 55Fe2-Tf was pH 7.4. 5) The uptake and reduction of iron from 55Fe2-Tf-Sepharose was inhibited by ferric citrate and by transition metals. We conclude that both Tf-independent and non-RME, Tf-dependent iron uptake proceed via a common redox system for iron. The mechanisms of cellular iron uptake can be separately evaluated in fibroblasts using 55Fe-citrate, 55Fe2-Tf, and 55Fe2-Tf-Sepharose beads.
结合了55Fe-转铁蛋白（Tf）的琼脂糖珠用于评估不采用受体介导的内吞作用（RME）的Tf依赖性铁摄取。55Fe2-Tf-琼脂糖的铁以时间和浓度依赖性方式被培养的人成纤维细胞还原并摄取（Km 7 microM；Vmax 128 pmol/mg/分钟）。该氧化还原系统在几个方面类似于非Tf依赖性铁摄取（Tf-IU，使用55Fe-柠檬酸盐评估）。1）NH4Cl不抑制从55Fe-柠檬酸盐和55Fe2-Tf-琼脂糖摄取铁，但抑制从55Fe2-Tf（RME系统）摄取铁。2）从55Fe2-Tf-琼脂糖和55Fe-柠檬酸盐摄取和还原铁随温度呈双曲线增加，这与RME摄取的S形曲线不同。3）来自55Fe-柠檬酸盐和55Fe2-Tf-琼脂糖的铁的亚细胞分布彼此相似，并且与55Fe2-Tf的不同。4）使用55Fe2-Tf-琼脂糖或55Fe-柠檬酸盐还原和摄取铁的最佳pH小于5.5，而从55Fe2-Tf摄取铁的最佳pH为7.4。5）柠檬酸铁和过渡金属抑制从55Fe2-Tf-琼脂糖摄取和还原铁。我们得出结论，非Tf依赖性和非RME的Tf依赖性铁摄取均通过铁的共同氧化还原系统进行。可以使用55Fe-柠檬酸盐、55Fe2-Tf和55Fe2-Tf-琼脂糖珠在成纤维细胞中分别评估细胞铁摄取的机制。
