Assessing Cytochrome-c Release from Mitochondria
In recent years substantial evidence has accumulated demonstrating a central regulatory role for mitochondria in cell death (1). More recently, mechanisms of cell death, through apoptosis, have been elucidated and increasingly point toward mitochondria as the gatekeepers of apoptosis. The morphological changes that define apoptosis can be attributed to the actions of a family of cysteinyl-dependent aspartate specific proteinases, called caspases (1). Members of this highly conserved protein family are expressed by all metazoan cells as zymogen proforms, which become activated by specific cleavage events that are usually mediated by other caspases. That is, activated caspases can cleave and activate other caspases, creating a cascade. The first caspases to become activated in this enzymatic cascade are called apical, or initiator caspases, and contain large prodomains that facilitate their recruitment to specialized macromolecular complexes. Once recruited to these complexes, apical caspases become activated and this can quickly lead to death of the cell through apoptosis. Therefore, the formation and regulation of these complexes are carefully regulated. Of the two apical caspase cascades identified thus far, mitochondria play a central role in one pathway and an amplification role in the other.
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