Thiol/disulfide interconversion in bovine lens aldose reductase induced byintermediates of glutathione turnover

The effectiveness of cysteine and cysteinylglycine to act as protein thiola ting agents was investigated using bovine lens aldose reductase (ALR2) as t he protein target. Disulfides of both thiol compounds appear to be very eff ective as ALR2 thiolating agents. Cysteine- and CysGly-modified ALR2 forms (Cys-ALR2 and CysGly-ALR2, respectively) are characterized by the presence of a mixed disulfide bond involving Cys298, as demonstrated by a combined e lectrospray mass spectrometry and Edman degradation approach. Both Cys-ALR2 and CysGly-ALR2 essentially retain the ability to reduce glyceraldehyde bu t lose the susceptibility to inhibition by Sorbinil and other ALR2 inhibito rs. Cys-ALR2 and CysGly-ALR2 are easily reduced back to the native enzyme f orm by dithiothreitol and GSH treatment; on the contrary, Cys and 2-mercapt oethanol appear to act as protein trans-thiolating agents, rather than redu cing agents. The treatment at 37 degreesC of both Cys-ALR2 and CysGly-ALR2, unlikely what observed for glutathionyl-modified ALR2 (GS-ALR2), promotes the generation of an intramolecular disulfide bond between Cys298 and Cys30 3 residues. A rationale for the special susceptibility of Cys-ALR2 and CysG ly-ALR2, as compared to GS-ALR2, to the thermally induced intramolecular re arrangement is given on the basis of a molecular dynamic and energy minimiz ation approach. A pathway of thiol/disulfide interconversion for bovine len s ALR2 induced, in oxidative conditions, by physiological thiol compounds i s proposed.