Involvement of calpain in postmortem proteolysis in the rat brain.
Calpain, a Ca(2+)-dependent neutral protease was examined to investigate its involvement in postmortem proteolysis in the rat brain. Western blotting analysis showed that the 240 kDa alpha-subunit of fodrin, a well-known substrate for calpain, was degraded to generate 150 kDa and 145 kDa fragments in the postmortem interval (0-24 h) at 25 +/- 3 degrees C. Postmortem proteolysis was dependent on ambient temperature. In in vitro experiments, the 150 kDa and 145 kDa fragments appeared in the homogenate with addition of Ca2+ (1 microM-1 mM) or in the microsomal fraction by incubation with purified calpain. Both calpain inhibitor-1 and leupeptin suppressed in vitro proteolysis. During the initial 0-24 h postmortem, the activity of m-calpain in the brain remained unaltered, while that of its endogenous inhibitor, calpastatin, decreased with the postmortem interval. These results indicate that calpain is involved in fodrin proteolysis in the postmortem rat brain. The ratio of the amount of the 150 kDa proteolytic product to that of the 240 kDa fodrin alpha-subunit was correlated significantly with the postmortem interval (0-16 h; r = 0.745).
Sorimachi
Y
Y
Department of Legal Medicine, Kyoto Prefectural University of Medicine, Japan.
Harada
K
K
Yoshida
K
K
D000818:Animals; D015153:Blotting, Western; D001923:Brain Chemistry; D002135:Calcium-Binding Proteins / Q000032:analysis; D002154:Calpain / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism*; D005554:Forensic Medicine; D008297:Male; D008840:Microfilament Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011180:Postmortem Changes; D051381:Rats; D017208:Rats, Wistar; D013696:Temperature; D013997:Time Factors; D013049:Spectrin
D002135:Calcium-Binding Proteins; D002154:Calpain; D002352:Carrier Proteins; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D013049:Spectrin; C033668:calpastatin