Flow cytometric measurement of calpain activity in living cells.
BACKGROUND
Calpains are intracellular, calcium-sensitive, neutral cysteine proteases that play crucial roles in many physiological and pathological processes. Calpain regulation is complex and activity is poorly correlated with calpain protein levels. Therefore a full understanding of calpain function requires robust methods for measuring activity.


METHODS
We describe and characterize a flow cytometric method for measuring calpain activity in live cells. This method uses the BOC-LM-CMAC reagent that readily diffuses into cells where it reacts with free thiols to enhance retention.


RESULTS
We show that the reagent is cleaved specifically by calpains and follows saturation kinetics. We use the assay to measure calpain activation following PDGF stimulation of rat fibroblasts. We also show that the calpain inhibitor PD150606 inhibits calpain with a K(i) of 12.5 muM and show that Mek inhibitors PD89059 and U0126 also suppress calpain activity. We also show that the assay can measure calpain activity in subpopulations of cells present in unfractionated cord blood or in HL60 human myelomonocytic leukemia cells.


CONCLUSIONS
Taken together, these experiments demonstrate that this assay is a reliable and useful method for measuring calpain activity in multiple cell types.
Niapour
Maryam
M
Arthritis and Immune Disorder Research Centre, University Health Network, Toronto, Ontario, Canada M5G 1L7.
Berger
Stuart
S
D000179:Acrylates / Q000494:pharmacology; D000818:Animals; D002154:Calpain / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002478:Cells, Cultured; D003374:Coumarins / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005312:Fetal Blood / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D051381:Rats; D013379:Substrate Specificity; D013696:Temperature
C453681:7-amino-4-chloromethylcoumarin; D000179:Acrylates; D003374:Coumarins; D004151:Dipeptides; C100442:PD 150606; D010982:Platelet-Derived Growth Factor; C522288:t-BOC-leucine-methionine amide-CMAC; D002154:Calpain