Isolation, characterization, and functional studies of rat liver iron regulatory protein 1

被引:4
作者
Eisenstein, RS
Barton, HA
Pettingell, WH
Bomford, AB
机构
[1] MIT,DIV TOXICOL,CAMBRIDGE,MA 02139
[2] TUFTS UNIV,USDA,HUMAN NUTR RES CTR AGING,BOSTON,MA 02111
关键词
iron regulatory protein 1; ferritin; translational control; iron metabolism;
D O I
10.1006/abbi.1997.0144
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Ferritin mRNAs are translationally regulated by the binding of either of two cytosolic proteins, iron regulatory protein 1 (IRP1) or IRP2, to the iron responsive element (IRE) located in their 5' untranslated region (UTR), Rat Liver IRP1 was purified by anion exchange, gel filtration, and affinity chromatography using a concatemerized version of the IRE. Two bands with M-r of 95,000 and 100,000 were observed by reducing SDS-PAGE, A single protein was responsible for both bands since: (1) [P-32]IRE RNA specifically cross-linked to both components; (2) alkylation with iodoacetamide resulted in formation of a single species with M-r of 95,000; and (3) they possessed identical peptide patterns after digestion with cyanogen bromide, The N-terminal sequence of rat liver IRP1 was MKNPFAHLAEPLDPAQPGKKFFNLNKLEDSRYGRLPFXIRVLLEAAV which is identical to the sequence deduced from the cDNA Rat liver IRP1 has an amino acid composition similar to that of bovine liver c-aconitase, Several species of IRP1 were observed by two-dimensional gel electrophoresis with pIs ranging from 7.5 to 8.0, Rat liver IRP1 bound the IRE with high affinity (K-D = 0.04 nM) and repressed translation of ferritin mRNA in vitro, IRP1 bound 100-fold less well to an IRE variant and failed to significantly repress translation of a ferritin mRNA containing the mutated IRE, We conclude that decreases in the affinity of interaction between IRP1 and the IRE, of a magnitude similar to that observed when the binding protein in converted to c-aconitase, are sufficient to significantly enhance translation of ferritin mRNA in vitro. (C) 1997 Academic Press
引用
收藏
页码:81 / 91
页数:11
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