THE DNA-BINDING OF PURIFIED AH RECEPTOR HETERODIMER IS REGULATED BY REDOX CONDITIONS

The Ah receptor from rat liver has been purified, using a specific oli gonucleotide affinity column, in order to characterize the components of the receptor and to investigate features that modulate its DNA-bind ing activity. The purified DNA-binding form of rat Ah receptor contain s three major components, with estimated molecular masses of 108, 98, and 96 kDa. Antibodies to two peptides from the mouse Ah receptor bind the 108-kDa protein, but not the 98-kDa protein, and bind weakly at t he position of the 96-kDa protein. The sequences of four peptides from samples containing both the 96- and 98-kDa proteins are all highly si milar to segments of the human Ah receptor nuclear translocator (Arnt) protein. Antibodies to a peptide from the human Arnt protein bind the 96- and 98-kDa proteins, but not the 108-kDa protein. These data show that the Ah receptor itself and two forms of the Amt protein are the major components of the purified DNA-binding form of receptor. In gel shift assays the purified receptor forms two specifically bound comple xes with a xenobiotic responsive element (XRE), which may correspond t o Ah receptor heterodimers with either of the two forms of Arnt protei n. The DNA binding of the purified heterodimer is substantially decrea sed under oxidizing conditions, Oxidation inhibits receptor DNA bindin g without greatly altering the size of the purified heterodimer, This sediments at 5.9S in its reduced form and at 6.5S in its oxidized form . Dithiothreitol restores the XRE binding of oxidized receptor, with s imilar effects on both of the receptor-XRE complexes. In the presence of nuclear extract, reduced thioredoxin also restores the XRE: binding of oxidized receptor. (C) 1995 Academic Press, Inc.