Purification of mouse MEP-1, a nuclear protein which binds to the metal regulatory elements of genes encoding metallothionein.
Metal regulatory elements (MREs) shared by metallothionein (MT) gene promoters are essential for metal induction of MT genes. MEP-1, a nuclear protein which binds to these elements has been purified from heavy metal-resistant mouse L cells using footprinting, Southwestern and UV cross-linking techniques to assay its binding activity. The purification scheme, starting from crude nuclear extracts, involved a combination of heparin-Sepharose and MRE-DNA affinity chromatography. The purified protein preparation showed a single polypeptide band of 108 kDa on polyacrylamide gel electrophoresis, and 2D-gel analyses revealed the presence of a protein species migrating as a single population of approximately 110 kDa. MEP-1 does not appear to be glycosylated since it eluted with the flow-through on a Wheat Germ Sepharose column. It was retained by a zinc-Chelating Sepharose column suggesting that amino acid residues (i.e., cysteine, histidine) which have an affinity for zinc ions are exposed on the protein surface. Binding studies with the purified protein indicated that it binds specifically to MRE sequences and that the binding can be abolished by a point mutation in the MRE core consensus sequence or by the addition of the chelating agent 1,10-phenanthroline. Binding activity can be restored by the addition of zinc ions to the chelated protein. These results suggest that MEP-1 is one of the major proteins interacting with MRE sequences.
Labbé
S
S
Centre de Recherche en Cancérologie, Université Laval, Hôtel-Dieu de Québec, Canada.
Larouche
L
L
Mailhot
D
D
Séguin
C
C
D000818:Animals; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D007739:L Cells; D008668:Metallothionein / Q000235:genetics*; D051379:Mice; D009687:Nuclear Proteins / Q000302:isolation & purification*; D010618:Phenanthrolines / Q000494:pharmacology; D012045:Regulatory Sequences, Nucleic Acid; D015032:Zinc / Q000494:pharmacology
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010618:Phenanthrolines; D008668:Metallothionein; D015032:Zinc; C025205:1,10-phenanthroline