OXIDATIVE MODIFICATION OF ALDOSE REDUCTASE INDUCED BY COPPER-ION - FACTORS AND CONDITIONS AFFECTING THE PROCESS

Bovine lens aldose reductase (ALR2) is inactivated by copper ion [Cu(I I)] through an oxygen-independent oxidative modification process. A st oichiometry of 2 equiv of Cu(II)/enzyme mol is required to induce inac tivation. While metal chelators such as EDTA or o-phenantroline preven t but do not reverse the ALR2 inactivation, DTT allows the enzyme acti vity to be rescued by inducing the recovery of the native enzyme form. The inactive enzyme form is characterized by the presence of 2 equiv of bound copper, at least one of which present as Cu(I), and by the pr esence of two lesser equivalents, with respect to the native enzyme, o f reduced thiol residues. Data are presented which indicate that the C u-induced protein modification responsible for the inactivation of ALR 2 is the generation on the enzyme of an intramolecular disulfide bond. GSH significantly interferes with the Cu-dependent inactivation of AL R2 and induces, through its oxidation to GSSG, the generation of an en zyme form linked to a glutathionyl residue by a disulfide bond.