Short peptide tools for monitoring caspase and proteasome activities in embryonal and adult rat brain lysates: an approach for the differential identification of proteases.
The numerous caspase-like activities present in nervous tissue can be investigated with labelled peptides. However, the cross-reactivities of peptides with both proteasomes and caspases complicate the analysis of protease activity. The pharmacological features of substrates and inhibitors specific for either caspases or proteasome caspase-like proteases in rat brain lysates were similar or identical to the profiles of commercially purified proteasome preparations. Caspase inhibitors bind directly to active proteasome centres, thus competing with selective antagonists of proteasomes. Separation of lysates by molecular weight does not separate active caspases from proteasomes because these enzymes co-localize under native electrophoresis. The addition of ATP or its analogues is associated with the differential modulation of proteasomal activity, which also leads to ambiguity in the data. However, induced caspase activity could be successfully differentiated from proteasome activity in embryonal brain lysates with the non-selective caspase inhibitors Z-VAD-FMK and Q-VD-OPh and the proteasome inhibitor AdaAhx(3)L(3)VS that are not cross-reactive. This strategy is proposed for the simultaneous examination of caspases and proteasomes using proteolysis experiments. The present study reveals that all of the caspase-like activities in the tissue lysates of non-injured adult rat brains were related to proteasomal caspase-like activities.
Laboratory of stem cell biology, A. Bogomoletz Institute of Physiology, National Academy of Sciences of Ukraine, Bogomoletz str., 4, 01024, Kiev, Ukraine. malysh@biph.kiev.ua
Igor M
IM
Prudnikov
Anton N
AN
Smirnov
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D015153:Blotting, Western; D001921:Brain / Q000187:drug effects / Q000196:embryology / Q000201:enzymology* / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism*; D005260:Female; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D066298:In Vitro Techniques; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D008297:Male; D010447:Peptide Hydrolases / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D000255:Adenosine Triphosphate; D010447:Peptide Hydrolases; D020169:Caspases; D046988:Proteasome Endopeptidase Complex; C001277:geldanamycin