title: Flow cytometric measurement of calpain activity in living cells.
abstract: 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.
authors: Arthritis and Immune Disorder Research Centre, University Health Network, Toronto, Ontario, Canada M5G 1L7.
authors: Maryam
authors: M
authors: Niapour
authors: Stuart
authors: S
authors: Berger
mesh_terms: 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
chemical_list: 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