Garrick L M, Dolan K G, Romano M A, Garrick M D
Department of Biochemistry, SUNY at Buffalo, New York 14214-3000, USA.
J Cell Physiol. 1999 Mar;178(3):349-58. doi: 10.1002/(SICI)1097-4652(199903)178:3<349::AID-JCP9>3.0.CO;2-R.
Belgrade (b) rats have an autosomal recessive, microcytic, hypochromic anemia. Transferrin (Tf)-dependent iron uptake is defective because of a mutation in DMT1 (Nramp2), blocking endosomal iron efflux. This experiment of nature permits the present study to address whether the mutation also affects non-Tf-bound iron (NTBI) uptake and to use NTBI uptake compared to Tf-Fe utilization to increase understanding of the phenotype of the b mutation. The distribution of 59Fe2+ into intact erythroid cells and cytosolic, stromal, heme, and nonheme fractions was different after NTBI uptake vs. Tf-Fe uptake, with the former exhibiting less iron into heme but more into stromal and nonheme fractions. Both reticulocytes and erythrocytes exhibit NTBI uptake. Only reticulocytes had heme incorporation after NTBI uptake. Properly normalized, incorporation into b/b heme was approximately 20% of +/b, a decrease similar to that for Tf-Fe utilization. NTBI uptake into heme was inhibited by bafilomycin A1, concanamycin, NH4Cl, or chloroquine, consistent with the endosomal location of the transporter; cellular uptake was uninhibited. NTBI uptake was unaffected after removal of Tf receptors by Pronase or depletion of endogenous Tf. Concentration dependence revealed that NTBI uptake into cells, cytosol, stroma, and the nonheme fraction had an apparent low affinity for iron; heme incorporation behaved like a high-affinity process, as did an expression assay for DMT1. DMT1 serves in both apparent high-affinity NTBI membrane transport and the exit of iron from the endosome during Tf delivery of iron in rat reticulocytes; the low-affinity membrane transporter, however, exhibits little dependence on DMT1.
贝尔格莱德（b）大鼠患有常染色体隐性、小细胞低色素性贫血。由于二价金属离子转运体1（DMT1，又名Nramp2）发生突变，转铁蛋白（Tf）依赖性铁摄取存在缺陷，该突变会阻碍内体铁外流。这种自然实验使得本研究能够探讨该突变是否也会影响非转铁蛋白结合铁（NTBI）的摄取，并通过比较NTBI摄取与Tf-Fe利用情况，来增进对b突变表型的理解。与Tf-Fe摄取相比，NTBI摄取后，59Fe2+在完整红细胞以及胞质、基质、血红素和非血红素组分中的分布有所不同，前者进入血红素的铁较少，但进入基质和非血红素组分的铁较多。网织红细胞和红细胞均表现出NTBI摄取。NTBI摄取后，只有网织红细胞有血红素掺入。经过适当标准化后，b/b型血红素的掺入量约为+/b型的20%，下降幅度与Tf-Fe利用情况相似。巴弗洛霉素A1、 concanamycin、NH4Cl或氯喹可抑制NTBI摄取进入血红素，这与转运体在内体中的定位一致；而细胞摄取不受抑制。用链霉蛋白酶去除Tf受体或耗尽内源性Tf后，NTBI摄取不受影响。浓度依赖性研究表明，NTBI摄取进入细胞、胞质、基质和非血红素组分对铁的亲和力明显较低；血红素掺入表现为高亲和力过程，DMT1的表达分析也是如此。在大鼠网织红细胞中，Tf传递铁的过程中，DMT1既参与了明显高亲和力的NTBI膜转运，也参与了铁从内体的外流；然而，低亲和力膜转运体对DMT1的依赖性较小。
