INVOLVEMENT OF CALPAIN IN POSTMORTEM PROTEOLYSIS IN THE RAT-BRAIN

Calpain, a Ca2+-dependent neutral protease was examined to investigate its involvement in postmortem proteolysis in the rat brain. Western b lotting analysis showed that the 240 kDa alpha-subunit of fodrin, a we ll-known substrate for calpain, was degraded to generate 150 kDa and 1 45 kDa fragments in the postmortem interval (0-24 h) at 25 +/- 3 degre es C. Postmortem proteolysis was dependent on ambient temperature. In in vitro experiments, the 150 kDa and 145 kDa fragments appeared in th e homogenate with addition of Ca2+ (1 mu M-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 0-2 4 h postmortem, the activity of m-calpain in the brain remained unalte red, while that of its endogenous inhibitor, calpastatin, decreased wi th the postmortem interval. These results indicate that calpain is inv olved 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 f odrin alpha-subunit was correlated significantly with the postmortem i nterval (0-16 h; r = 0.745).