Abstract
Calpains are cysteine proteases involved in a number of physiological and pathological processes, yet our knowledge of substrates cleaved in vivo, in intact cells, is scarce. In this work we made an attempt to develop a technique for finding calpain substrates in intact Drosophila Schneider S2 cells. The procedure consists in comparative 2D gelelectrophoresis: three identical samples were treated in different ways: A (control, no addition), B, activated (Ca 2+ and ionomycin added), C, inactivated (additions as in B + specific calpain inhibitor). 2D gel pattern were analyzed by densitometry. Spots showing density relation A > B << C were identified by mass spectroscopy. In a typical run, 11 candidate substrates were recognized; out of these, four were randomly selected: all four were verified to be calpain substrates, by digestion of the recombinant protein with recombinant calpain.
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