Lp82 calpain during rat lens maturation and cataract formation.
OBJECTIVE
To measure changes in levels of Lp82 during maturation and selenite cataract formation in rat lens. Lp82 is a lens-specific, calcium-activated isozyme from the calpain family of cysteine proteases (EC 34.22.17).


METHODS
Competitive RT-PCR was used to assess Lp82 and m-calpain mRNA concentrations. Immunoblotting and ELISA after DEAE chromatography measured Lp82 and m-calpain protein levels. Casein zymography assessed proteolytic activities in regions and whole lenses from maturing rats.


RESULTS
Levels of Lp82 mRNA, protein, and caseinolytic activity decreased more rapidly during maturation of rat lens than for m-calpain. Unexpectedly, the water-insoluble fraction of rat lens contained enzymatically active Lp82. Selenite injection also caused major loss of Lp82 protein during cataract formation.


CONCLUSIONS
Lp82 is a proteolytic enzyme likely functioning in early lens development and maturation. The rapid loss of Lp82 activity during lens maturation is probably caused by three factors: autodegradation associated with the proteolysis of soluble and insoluble proteins occurring in the rat lens nucleus, association of Lp82 with the lens insoluble fraction, and loss of Lp82 mRNA. Lp82 may function early in lens maturation along with m-calpain, which then is predominant in the latter stages of maturation. Proteolysis in selenite cataract is partially caused by over-activation of Lp82.
Shearer
T R
TR
Department of Oral Molecular Biology, Oregon Health Sciences University, Portland 97201, USA. shearert@ohsu.edu
Ma
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Shih
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Hata
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Fukiage
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Nakamura
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Azuma
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D000375:Aging / Q000502:physiology; D000818:Animals; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism; D002386:Cataract / Q000139:chemically induced / Q000201:enzymology*; D002848:Chromatography, DEAE-Cellulose; D004797:Enzyme-Linked Immunosorbent Assay; D015151:Immunoblotting; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007908:Lens, Crystalline / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018038:Sodium Selenite
D002364:Caseins; D007527:Isoenzymes; D012333:RNA, Messenger; D002154:Calpain; D018038:Sodium Selenite