Effect of pH, ionic charge, and osmolality on cytochrome c-mediated caspase-3 activity.
Cytochrome c-mediated activation of caspase-3 is the final common pathway for most signals that induce apoptosis. Before release of cytochrome c from mitochondria, K(+) and Cl(-) efflux and intracellular acidification must occur. We have utilized an in vitro assay to examine the role of pH, cations, anions, and uncharged molecules on the process of cytochrome c-mediated activation of procaspase-3. In this cell-free system, a pH above 7.4 severely suppressed the activation of procaspase-3 but not the activity of caspase-3. KCl, NaCl, and other salts all inhibited caspase activation, but uncharged molecules did not. Comparison of the inhibitory capacity of various salts suggests that the crucial element in causing suppression is the cation. The inhibition of alkaline pH could be overcome by increasing concentrations of cytochrome c, whereas the inhibition of ionic charge could not, suggesting that pH and salts affect the activation of caspase-3 by different mechanisms.
Segal
M S
MS
Division of Nephrology, Hypertension and Transplantation, Department of Medicine, University of Florida, Gainesville, Florida 32610, USA. segalms@medicine.ufl.edu
Beem
E
E
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002412:Cations / Q000378:metabolism; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology* / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000166:cytology; D009842:Oligopeptides / Q000494:pharmacology; D009994:Osmolar Concentration; D011189:Potassium Chloride / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D014882:Water-Electrolyte Balance / Q000187:drug effects / Q000502:physiology*
D002412:Cations; D015853:Cysteine Proteinase Inhibitors; D003574:Cytochrome c Group; D009842:Oligopeptides; C120992:benzyloxycarbonyl aspartyl-glutamyl-valyl-aspartyl-chloromethyl ketone; D012965:Sodium Chloride; D011189:Potassium Chloride; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases