Abstract
Release of cytochrome c from mitochondria triggers activation of caspase proteases and death of a cell by apoptosis. However, the mechanism and kinetics of cytochrome c release remain unknown. Here we study this event by using green fluorescent protein (GFP)-tagged cytochrome c, and find that the release of cytochrome-c-GFP always precedes exposure of phosphatidylserine and the loss of plasma-membrane integrity - characteristics of apoptotic cells. Once initiated, the release of cytochrome- c-GFP continues until all of the protein is released from all mitochondria in individual cells, within about 5 minutes, regardless of the type or strength of stimulus or the time elapsed since the stimulus was applied. Temperatures ranging from 24 degrees C to 37 degrees C do not change the duration of release, and nor does the addition of caspase inhibitors. Further, we find that the electron-transport chain can maintain the mitochondrial transmembrane potential even after cytochrome c has been released.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adenosine Triphosphate / metabolism
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Amino Acid Chloromethyl Ketones / pharmacology
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Apoptosis*
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Caspase Inhibitors
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Caspases / metabolism
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Cysteine Proteinase Inhibitors / pharmacology
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Cytochrome c Group / genetics
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Cytochrome c Group / metabolism*
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Digitonin / pharmacology
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Electron Transport / drug effects
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Electron Transport / radiation effects
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Enzyme Activation / drug effects
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Enzyme Activation / radiation effects
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Enzyme Inhibitors / pharmacology
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Green Fluorescent Proteins
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HeLa Cells
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Humans
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Image Processing, Computer-Assisted
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Intracellular Membranes / drug effects
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Intracellular Membranes / radiation effects
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Luminescent Proteins / genetics
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Membrane Potentials / drug effects
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Mitochondria / metabolism
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Oligomycins / pharmacology
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Phosphatidylserines / metabolism
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Sodium Azide / pharmacology
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Temperature
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Ultraviolet Rays
Substances
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Amino Acid Chloromethyl Ketones
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Caspase Inhibitors
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Cysteine Proteinase Inhibitors
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Cytochrome c Group
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Enzyme Inhibitors
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Luminescent Proteins
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Oligomycins
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Phosphatidylserines
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Recombinant Fusion Proteins
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benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone
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Green Fluorescent Proteins
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Adenosine Triphosphate
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Sodium Azide
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Caspases
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Digitonin