title: A Northwestern blotting approach for studying iron regulatory element-binding proteins.
abstract: At least two proteins binding to iron regulatory elements (IRE) in mRNA are known, designated as iron regulatory proteins (IRP) 1 and 2. Their binding activity is widely studied by electrophoretic mobility shift assays (EMSA), which resolves one or two bands depending on the species. We used Northwestern blotting to resolve this EMSA complex into four components, and identified two other IRE-binding peptides present in HepG2 cell extracts. We designate these six peptide bands A to F on Northwestern blots, ranging in apparent molecular weight from 111 to 37 kDa. Band C is lost when cells are preloaded with iron or when leupeptin (but not several other protease inhibitors) is included in the extraction buffer. Band E is also lost with leupeptin but increases with iron loading. Binding of all bands is sensitive to iron in vitro. Two-dimensional electrophoresis reveals additional processing, especially indicating charge variants of band C. Northwestern bands A and B both react with an antibody to IRP-1 on parallel Western blots. We conclude that cellular processing can produce multiple IRE-binding species that may be involved in a more complex regulation of iron metabolism than generally appreciated. The Northwestern approach should facilitate studies of processing and binding requirements of proteins and peptides that recognize the IRE sequence.
authors.affiliation: Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.
authors.forename: Zvezdana
authors.initials: Z
authors.lastname: Popovic
authors.forename: Douglas M
authors.initials: DM
authors.lastname: Templeton
mesh_terms: D015152:Blotting, Northern; D015153:Blotting, Western; D045744:Cell Line, Tumor; D006801:Humans; D035941:Iron Regulatory Protein 1 / Q000378:metabolism*; D035942:Iron Regulatory Protein 2 / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D038621:Regulatory Sequences, Ribonucleic Acid / Q000502:physiology*
chemical_list: D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D038621:Regulatory Sequences, Ribonucleic Acid; D035941:Iron Regulatory Protein 1; D035942:Iron Regulatory Protein 2
references.citation: Arch Biochem Biophys. 1995 Jan 10;316(1):128-34
references.pmid: 7840606
references.citation: Trends Biochem Sci. 2000 Aug;25(8):352-6
references.pmid: 10916152
references.citation: Cell. 1991 Mar 8;64(5):881-3
references.pmid: 2001588
references.citation: Nucleic Acids Res. 1995 Jul 25;23(14):2579-83
references.pmid: 7544459
references.citation: Blood. 1999 Sep 15;94(6):2128-34
references.pmid: 10477743
references.citation: J Biol Chem. 2001 Jul 20;276(29):27685-92
references.pmid: 11313346
references.citation: Blood. 1996 Apr 1;87(7):2983-92
references.pmid: 8639920
references.citation: J Biol Chem. 2002 Nov 22;277(47):45518-28
references.pmid: 12198135
references.citation: Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7536-40
references.pmid: 1502165
references.citation: FEBS Lett. 2000 Nov 17;485(1):25-8
references.pmid: 11086159
references.citation: Mol Cell Biochem. 2004 Oct;265(1-2):37-45
references.pmid: 15543932
references.citation: J Biol Chem. 1994 Dec 9;269(49):30904-10
references.pmid: 7983023
references.citation: EMBO J. 1995 Jun 15;14(12):2917-24
references.pmid: 7796817
references.citation: J Biol Chem. 1995 Sep 15;270(37):21645-51
references.pmid: 7665579
references.citation: EMBO J. 2000 Nov 1;19(21):5692-700
references.pmid: 11060020
references.citation: J Virol. 1996 Apr;70(4):2184-90
references.pmid: 8642641
references.citation: J Biol Chem. 2003 Feb 21;278(8):5710-7
references.pmid: 12468554
references.citation: Hepatology. 1995 Sep;22(3):828-32
references.pmid: 7657289
references.citation: J Biol Chem. 1996 Mar 22;271(12):7168-76
references.pmid: 8636154
references.citation: Arch Biochem Biophys. 1994 Jun;311(2):517-22
references.pmid: 8203918
references.citation: Nature. 2003 Aug 21;424(6951):961-5
references.pmid: 12931193
references.citation: Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):574-8
references.pmid: 8290565
references.citation: J Cell Physiol. 2002 Feb;190(2):218-26
references.pmid: 11807826
references.citation: Eur J Biochem. 1993 Dec 1;218(2):657-67
references.pmid: 8269957
references.citation: EMBO J. 1999 Nov 1;18(21):6073-83
references.pmid: 10545118
references.citation: Annu Rev Nutr. 2000;20:627-62
references.pmid: 10940348
references.citation: J Biol Chem. 1999 Feb 19;274(8):5047-52
references.pmid: 9988751
references.citation: Chem Rev. 1996 Nov 7;96(7):2335-2374
references.pmid: 11848830
references.citation: Nucleic Acids Res. 1994 Jul 11;22(13):2627-33
references.pmid: 7518918
references.citation: Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11730-4
references.pmid: 1334546
references.citation: J Biol Chem. 1993 Dec 25;268(36):27363-70
references.pmid: 8262977
references.citation: Proc Natl Acad Sci U S A. 1998 Apr 28;95(9):4924-8
references.pmid: 9560204
references.citation: Biochemistry. 1997 Apr 1;36(13):3950-8
references.pmid: 9092825
references.citation: J Biol Chem. 1998 Jan 16;273(3):1821-4
references.pmid: 9430733
references.citation: Blood. 1999 Nov 1;94(9):3205-11
references.pmid: 10556209
references.citation: EMBO J. 1994 Jan 15;13(2):453-61
references.pmid: 7508861
references.citation: Biochemistry. 2004 Jan 13;43(1):195-204
references.pmid: 14705945
references.citation: J Biol Chem. 1995 Jul 14;270(28):16529-35
references.pmid: 7622457
references.citation: Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7321-5
references.pmid: 8041788
references.citation: Proc Natl Acad Sci U S A. 1988 Apr;85(7):2171-5
references.pmid: 3127826
references.citation: Nat Cell Biol. 2003 Apr;5(4):336-40
references.pmid: 12629548
references.citation: Cell. 1989 Jul 28;58(2):373-82
references.pmid: 2752428