Identifying calpain substrates in intact S2 cells of Drosophila |
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Authors: | Zoltan Bozoky Julia Dancsok Eva Klement Peter Friedrich |
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Affiliation: | a Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1518 Budapest, P.O. Box 7, Hungary b Proteomics Research Group, Biological Research Center, Hungarian Academy of Sciences, H-6726 Szeged, Temesvari krt. 62, Hungary c Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, USA |
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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 (Ca2+ 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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Keywords: | Calpain substrate In vivo detection Schneider cells Drosophila 2D-electrophoresis |
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