Activation of calpain in lens: a review and proposed mechanism.
The purpose of these experiments was to develop a hypothesis to explain activation of m-calpain in cataractogenesis observed in rodents. The in vitro model used to study m-calpain activation was to correlate breakdown of the 'reporter' protein alpha-crystallin with the appearance of activated m-calpain using protein sequencing and casein zymography. Incubation of alpha-crystallins with m-calpain and Ca2+ caused proteolysis of alpha-crystallins and accumulation of new polypeptides. E64 and calpain inhibitor I each inhibited proteolysis of alpha-crystallins. The N-terminus of the 80 kDa subunit of m-calpain was blocked at time 0 (pro calpain). After incubation with Ca2+, the remaining 80 kDa subunit of m-calpain gave a N-terminal sequence of KDREAAEGLG, indicating loss of nine amino acid from the N-terminus (autolysed calpain). The new 43 kDa m-calpain fragment also gave a N-terminal sequence of KDREAAEGLG, indicating the same loss of the first nine amino acids on the N-terminus as well as a major loss of the C-terminal half of the subunit (degraded calpain). In contrast, the N-terminus of the 80 kDa subunit of m-calpain remained blocked when E64 was present (unautolysed form). Moreover, the Ca2+ concentration required for proteolysis decreased when calpain was pre-incubated with Ca2+, although proteolysis of alpha-crystallin required a higher Ca2+ concentration than proteolysis of casein. These data suggested that the sequence of events for m-calpain activation were unautolysed, autolysed and finally degraded calpain. Unautolysed and/or autolysed calpains may be proteolytically active against alpha-crystallin.
Azuma
M
M
Department of Oral Molecular Biology, Oregon Health Sciences University, Portland 97201, USA.
Fukiage
C
C
David
L L
LL
Shearer
T R
TR
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003459:Crystallins / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006023:Glycoproteins / Q000494:pharmacology; D007908:Lens, Crystalline / Q000378:metabolism*; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008954:Models, Biological; D011817:Rabbits; D017421:Sequence Analysis
D003459:Crystallins; D015853:Cysteine Proteinase Inhibitors; D006023:Glycoproteins; C039304:calpain inhibitors; D002154:Calpain; D007930:Leucine; C024974:E 64; D002118:Calcium