Oxidation enhances calpain-induced turbidity in young rat lenses.
OBJECTIVE
To determine if oxidation enhances turbidity after proteolysis of rat lens crystallins by the calcium-activated protease calpain (EC 3.4.22.17).


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 were cultured for 6 days in low calcium medium and diamide. The lenses were then photographed to assess transmission of light. SDS-PAGE and immunoblotting assessed proteolysis of crystallins, alpha-spectrin, and activation of calpain.


RESULTS
Appreciable in vitro turbidity occurred in soluble proteins from young rat lenses after proteolysis of crystallins by endogenous calpain. Calpain inhibitor E64, or anti-oxidants DTE and GSH, inhibited this turbidity. On the other hand, the oxidant diamide markedly enhanced calpain-induced turbidity. Cultured rat lenses showed elevated intralenticular 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 proteolyzed. Oxidation may be one of the factors promoting light scatter and insolubilization after proteolysis. These data are consistent with the hypothesis that proteolysis of crystallins from young rat lens may expose cysteine residues, which are then oxidized, become insoluble and scatter light.
Nakamura
Y
Y
Research Laboratories, Senju Pharmaceutical Corporation Limited, Kobe, Japan.
Fukiage
C
C
Azuma
M
M
Shearer
T R
TR
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002386:Cataract / Q000139:chemically induced / Q000473:pathology; D003459:Crystallins / Q000378:metabolism; D046508:Culture Techniques; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003958:Diamide / Q000494:pharmacology; D004226:Dithioerythritol / Q000494:pharmacology; D004357:Drug Synergism; D005978:Glutathione / Q000494:pharmacology; D007908:Lens, Crystalline / Q000187:drug effects* / Q000473:pathology / Q000503:physiopathology*; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D010447:Peptide Hydrolases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D000975:Antioxidants; D003459:Crystallins; D015853:Cysteine Proteinase Inhibitors; D016877:Oxidants; D003958:Diamide; D004226:Dithioerythritol; D010447:Peptide Hydrolases; D002154:Calpain; D005978:Glutathione; D007930:Leucine; C024974:E 64