LIMITED PROTEOLYTIC PROCESSING OF THE MATURE FORM OF CATHEPSIN-D IN HUMAN AND MOUSE-BRAIN - POSTMORTEM STABILITY OF ENZYME STRUCTURE AND ACTIVITY

The mature form of cathepsin D (Cat D), purified to homogeneity from p ostmortem human brain or mouse brain, behaved as a 42-kDa protein in i ts native state but revealed additional proteolytic processing under d enaturing conditions. Human brain Cat D was composed of a 30-32 kDa he avy chain and a protein doublet consisting of 14 and 15 kDa light chai ns. Mouse Cat D, which closely resembled the human enzyme in amino aci d composition, existed mainly as the uncleaved 42-kDa protein, but up to 40% existed as a complex of 30-32 kDa and 12-14 kDa chains. The 3:1 ratio of light to heavy (30-32 kDa) chains suggested processing of so me 30-kDa chains. Cleavage of the 42-kDa chain could not be induced au tolytically. Human brain Cat D had a 2-3-fold higher specific activity than the mouse enzyme but shared other properties, including similar biphasic pH optima (peaks at pH 3.0 and 4.2), K-m values For methemogl obin and inhibitor profiles. Human Cat D displayed the same polypeptid e chain composition when purified from brains differing in postmortem interval (3-28 h). Fresh SH-SY5Y human neuroblastoma cells analyzed on Western blots with anti-Cat D antibodies also displayed only cleaved forms of mature Cat D. Furthermore, brain Cat D isolated from mice sto red after death for 5, 15 or 30 h at 25 degrees C contained the same m olar ratios of cleaved and uncleaved enzyme found in fresh mouse brain . Cat D activity was stable in human brains with postmortem intervals up to 27 h and stored frozen for up to 3 years. Similarly, total Cat D activity was essentially unchanged in brains of mice subjected to sim ulated postmortem conditions for 0.5-42 h, although 20% of the total s oluble brain protein became insoluble during this postmortem interval. These results demonstrate a remarkable postmortem stability of Cat D and strongly suggest that limited proteolytic cleavage of mature brain Cat D is an in vivo event, the extent of which varies markedly in dif ferent species.