Identifying calpain substrates in intact S2 cells of Drosophila.
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.
Bozoky
Zoltan
Z
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1518 Budapest, P.O. Box 7, Hungary.
Alexa
Anita
A
Dancsok
Julia
J
Gogl
Gergo
G
Klement
Eva
E
Medzihradszky
Katalin F
KF
Friedrich
Peter
P
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D017931:DNA Primers; D004330:Drosophila / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D013058:Mass Spectrometry; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D029721:Drosophila Proteins; D011994:Recombinant Proteins; D015759:Ionomycin; D002154:Calpain; D002118:Calcium