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Correlating the fractional inhibition of caspase-3 in NT2 cells with apoptotic markers using an active-caspase-3 enzyme-linked immunosorbent assay
- Source :
- Analytical Biochemistry. 350:32-40
- Publication Year :
- 2006
- Publisher :
- Elsevier BV, 2006.
-
Abstract
- A rapid and quantitative method for measuring the activity and fractional inhibition of enzymes within their natural cellular environment remains an unmet need in drug discovery. We describe the use of a nonradioactive quantitative enzyme-linked immunosorbent assay (ELISA) for measuring intracellular caspase activity that is amenable to robotic automation. The ELISA specifically detects active-caspase-3 and was used to correlate the in-cell activity of caspase-3 with the progress of caspase-3-mediated events under varying concentrations of caspase-3 inhibitors in NT2 cells. We examined the cleavage of endogenous substrates (poly(ADP-ribose)polymerase and αII-spectrin), the extent of DNA fragmentation, and the autocatalytic removal of the caspase-3 prodomain as markers of caspase-3 activity. To impart inhibition of the downstream markers, a greater level of caspase-3 inhibition was required. Although the functional markers were found not to accurately predict intracellular caspase-3 activity, we found that the inhibition of intracellular caspase-3 was highly correlated (R2 = 0.96) to the inhibition of DNA fragmentation. Also, by comparing the potency of the different inhibitors against the intracellular enzyme versus the purified enzyme, the effects of inhibitor functional groups on whole-cell activity were addressed.
- Subjects :
- medicine.medical_treatment
Biophysics
Biotin
Apoptosis
Enzyme-Linked Immunosorbent Assay
Caspase 3
DNA Fragmentation
Cysteine Proteinase Inhibitors
Poly(ADP-ribose) Polymerase Inhibitors
Sensitivity and Specificity
Biochemistry
Cell Line
Inhibitory Concentration 50
Coumarins
medicine
Humans
Molecular Biology
Caspase
Polymerase
chemistry.chemical_classification
Protease
biology
Spectrin
Dipeptides
Cell Biology
Caspase Inhibitors
Molecular biology
Enzyme
chemistry
Caspases
biology.protein
DNA fragmentation
Electrophoresis, Polyacrylamide Gel
Oligopeptides
Intracellular
Subjects
Details
- ISSN :
- 00032697
- Volume :
- 350
- Database :
- OpenAIRE
- Journal :
- Analytical Biochemistry
- Accession number :
- edsair.doi.dedup.....e8394dc1b7f101579ae5f7d517728de3