Protein for Lp82 calpain is expressed and enzymatically active in young rat lens.
mRNA for a newly discovered isoform of calpain, termed Lp82, was recently discovered in young rat lens. The purpose of the present experiments was to test for expression of Lp82 protein. Casein zymography after incubation with calcium was used to detect Lp82 proteolytic activity in regions of lenses from young rats. Lp82 protein was detected by immunoblotting or by ELISA after DEAE-5PW chromatography using a polyclonal antibody generated to a peptide sequence in Lp82. Northern blot analysis assessed expression of Lp82 mRNA. Four results demonstrated expression of Lp82 protein; (1) immunoblot reactivity at the predicted molecular mass of 82 kDa, (2) a unique band of calcium-activated lysis in casein zymograms, (3) partial purification and retention of activity from a single Lp82 peak on DEAE-5PW chromatography, and (4) positive immunoblotting and Northern blot analysis only in lens and not in other rat tissues. These results showed that Lp82 protein is lens-preferred, relatively abundant in young rats (especially nucleus), and enzymatically active. Proteolysis of crystallins in the nucleus of young rat lens during normal maturation and cataract formation, formerly attributed solely to m-calpain, may in fact be due to concerted action of both lens Lp82 and ubiquitous m-calpain.
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Department of Oral Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201, USA.
Shih
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Hata
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Fukiage
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Azuma
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Shearer
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D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002154:Calpain / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002364:Caseins; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007908:Lens, Crystalline / Q000201:enzymology*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar
D002364:Caseins; D007527:Isoenzymes; D012333:RNA, Messenger; D002154:Calpain