Resting membrane potential as a marker of apoptosis: studies on Xenopus oocytes microinjected with cytochrome c.
Observation of the electrical potential difference across the cell membrane is described as a new method for monitoring apoptosis of a single cell. The resting membrane potential (DeltaPsi) of Xenopus oocytes has been recorded in real time following microinjection of cytochrome c. Soon after microinjection, DeltaPsi becomes less negative and attains a new constant value with a half time, t(m), of about 35 (+ /- 5) min at all cytochrome c concentrations greater than 1 microM. The cytosol extract of cytochrome c-injected oocytes shows DEVD proteolytic activity characteristic of aspartate specific proteases, implicating an apoptotic death pathway. In response to the delivery of cytochrome c into the cytosol, caspases are activated within 7 min while the changes in DeltaPsi begin to occur after about 30 min. The method described here will be potentially useful to assess the effectiveness of cell death regulators and modulators of synthetic and biological origin, and the results presented shed light on the currently debated issue of the importance of the redox state of cytochrome c in the initiation of apoptosis.
Bhuyan
A K
AK
Centre for Biochemical Technology (CSIR), Delhi University Campus, Delhi 110 007, India.
Varshney
A
A
Mathew
M K
MK
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002462:Cell Membrane / Q000502:physiology; D003574:Cytochrome c Group / Q000008:administration & dosage* / Q000378:metabolism / Q000494:pharmacology; D045304:Cytochromes c; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008845:Microinjections; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010084:Oxidation-Reduction; D010447:Peptide Hydrolases / Q000378:metabolism; D011487:Protein Conformation; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D014982:Xenopus laevis
C014059:CYC1 protein, S cerevisiae; D061945:Caspase Inhibitors; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c; D010447:Peptide Hydrolases; D020169:Caspases; C108711:DEVDase