Oxidative Modification of Aldose Reductase Induced by Copper Ion. Factors and Conditions Affecting the Process † Ilaria Cecconi, Maria Moroni, Pier Giuseppe Vilardo, Massimo Dal Monte, Paola Borella, ‡ Giulio Rastelli, § Luca Costantino, § Donita Garland, | Deborah Carper, | J. Mark Petrash, ⊥ Antonella Del Corso, and Umberto Mura* Dipartimento di Fisiologia e Biochimica, UniVersita ` di Pisa, Via S. Maria, 55-56100 Pisa, Italy, Dipartimento di Scienze Biomediche, UniVersita ` di Modena, Via Campi, 287, 41100 Modena, Italy, Dipartimento di Scienze Farmaceutiche, UniVersita ` di Modena, Via Campi, 183, 41100 Modena, Italy, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892, and Departments of Ophthalmology and Visual Sciences and of Genetics, Washington UniVersity School of Medicine, St. Louis, Missouri 63110 ReceiVed May 18, 1998; ReVised Manuscript ReceiVed August 13, 1998 ABSTRACT: Bovine lens aldose reductase (ALR2) is inactivated by copper ion [Cu(II)] through an oxygen- independent oxidative modification process. A stoichiometry of 2 equiv of Cu(II)/enzyme mol is required to induce inactivation. While metal chelators such as EDTA or o-phenantroline prevent but do not reverse the ALR2 inactivation, DTT allows the enzyme activity 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 presence of two lesser equivalents, with respect to the native enzyme, of reduced thiol residues. Data are presented which indicate that the Cu-induced protein modification responsible for the inactivation of ALR2 is the generation on the enzyme of an intramolecular disulfide bond. GSH significantly interferes with the Cu-dependent inactivation of ALR2 and induces, through its oxidation to GSSG, the generation of an enzyme form linked to a glutathionyl residue by a disulfide bond. Copper is a trace element involved in the mechanism of action of several enzymes and other functional proteins (1, 2). In vivo, the level of the free metal is strictly controlled by chelating proteins, such as ceruloplasmin, transcuprein, and albumin, which are all devoted to storage and transport (1). There are, however, pathological conditions (3-7) in which the concentration of the metal ion increases and causes damage. Similar to iron, copper promotes the Fenton reaction and therefore may contribute to oxidative stress in biological systems (8-13). Moreover, due to its effective binding capability to polypeptides (14, 15) and nucleic acids (16-18), copper ion has the potential for site-directed action on these molecular targets, effectively interfering with normal cell function and proliferation (19). Different metal- catalyzed oxidation (MCO) 1 systems have been used in order to study the ability of copper to induce oxidative damage (20-22). In this study, aldose reductase (alditol:NADP + oxidoreductase, EC 1.1.1.21), purified from bovine lens was chosen as the protein target. Aldose reductase, which catalyzes the NADPH-dependent reduction of aldoses, as well as a variety of aliphatic and aromatic aldehydes to the corresponding alcohols (23), was shown to be especially susceptible to thiol-mediated oxidative modification induced by the oxygen radical generating system Fe 2+ /EDTA (24). Thus, different thiol compounds under oxidative conditions, lead in vitro to enzyme forms displaying altered kinetic and structural properties (25-28). Among these enzyme forms, the S-glutathionyl-modified ALR2, can be generated in vitro by treatment of both bovine lens and human ALR2 (27, 29) with GSH in the presence of Fe 2+ /EDTA or with GSSG. This enzyme form was detected in intact bovine lens subjected to hyperbaric oxygen treatment (30). In vitro studies demonstrate that the modi- fication of ALR2 appears to proceed by a preliminary oxidation of thiols to disulfides which would be the true modifying agents of the protein. The MCO system would act by generating or propagating oxygen radicals in the bulk solution without a significant direct interaction with the enzyme molecule, and the subsequent enzyme modification would be the result of the increasing concentration of GSSG formed as a consequence of the GSH scavenging action. When oxidative stress is induced by copper ion, GSH counteracts the stress by its well-known oxidant scavenging action as well as by chelating the copper, which would then be more easily transferred from the bulk solution to metal- binding proteins (31, 32). We describe here the special effectiveness of copper ion in inducing bovine lens ALR2 inactivation, either in the absence or in the presence of GSH, the most highly represented lens thiol. † This work was supported in part by the “Target Project on Cellular Oxidative Stress” of the Italian National Research Council and in part by Italian Board of Education (MURST). * To whom correspondence should be addressed at the Universita ` di Pisa. Tel: 39-50-500292. Fax: 39-50-502583. E-mail: cmario @dfb.unipi.it. ‡ Dipartimento di Scienze Biomediche, Universita ` di Modena. § Dipartimento di Scienze Farmaceutiche, Universita ` di Modena. | National Institutes of Health. ⊥ Washington University School of Medicine. 1 Abbreviations: MCO, metal catalyzed oxidation; ALR2, aldose reductase; BCDS, bathocuproinedisulphonic acid; DTNB, 5,5′-dithiobis- (2-nitrobenzoic acid); DTT, dithiothreitol; GS-ALR2, glutathione- modified ALR2; OP, o-phenantroline; SD, standard deviation of the mean; SOD, superoxide dismutase. 14167 Biochemistry 1998, 37, 14167-14174 S0006-2960(98)01159-3 CCC: $15.00 © 1998 American Chemical Society Published on Web 09/17/1998