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 p romoters are essential for metal induction of MT genes. MEP-1, a nucle ar 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 purificati on scheme, starting from crude nuclear extracts, involved a combinatio n of heparin-Sepharose and MRE-DNA affinity chromatography. The purifi ed protein preparation showed a single polypeptide band of 108 kDa on polyacrylamide gel electrophoresis, and 2D-gel analyses revealed the p resence of a protein species migrating as a single population of appro ximately 110 kDa. MEP-1 does not appear to be glycosylated since it el uted with the flow-through on a Wheat Germ Sepharose column. It was re tained 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 puri fied 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-phe nanthroline. 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.