Oxidation enhances calpain-induced turbidity in young rat lenses

Purpose. To determine if oxidation enhances turbidity after proteolysis of rat lens crystallins by the calcium-activated protease calpain (EC 3.4.22.1 7). Methods. Total soluble proteins from young rat lens were hydrolyzed for 24 hr by endogenous lens calpain, and the proteins were further incubated with the oxidant diamide for up to 7 days. Turbidity was measured daily at 405 nm. To measure proteolysis and turbidity in cultured lenses, rat lenses wer e cultured for 6 days in low calcium medium and diamide. The lenses were th en photographed to assess transmission of light. SDS-PAGE and immunoblottin g assessed proteolysis of crystallins, a-spectrin, and activation of calpai n. Results. Appreciable in vitro turbidity occurred in soluble proteins from y oung rat lenses after proteolysis of crystallins by endogenous calpain. Cal pain inhibitor E64, or anti-oxidants DTE and GSH, inhibited this turbidity. On the other hand, the oxidant diamide markedly enhanced calpain-induced t urbidity. Cultured rat lenses showed elevated intra-lenticular calcium and proteolysis of crystallins by calpain, but no nuclear cataract. Addition of diamide to the culture medium caused development of nuclear cataract. Conclusions. Diamide enhanced turbidity only when crystallins were proteoly zed. Oxidation may be one of the factors promoting light scatter and insolubiliz ation after proteolysis. These data are consistent with the hypothesis that proteolysis of crystallins from young rat lens may expose cysteine residue s, which are then oxidized, become insoluble and scatter light.