Abstract
Programmed cell death (PCD) is an irreversible, genetically-controlled form of cell suicide in which an endogenous biochemical pathway leads to morphological changes and ultimately, cellular demise. PCD is accompanied by de-novo protein synthesis of a family of proteases-"caspases" that are often used as a diagnostic marker of PCD. Although phytoplankton do not contain true caspases, caspase-like activity (hypothetical proteins with analogous activity) has been traditionally used as a diagnostic marker of PCD in marine phytoplankton. Increased caspase-like proteolytic activity was demonstrated when synthetic fluorogenic activity substrates specific for caspases (with an Asp at the P1 position) were applied upon PCD induction. Metacaspases, cysteine proteases, share structural properties with those of caspases, yet they are highly specific for Arg and Lys cleavage site at the P1 position implying that caspase specific substrates are not indicative of metacaspase catalytic activity. This method specifically tests direct metacaspase activity in phytoplankton by the cleavage of the fluorogenic metacaspase substrate Ac-VRPR-AMC. Metacaspase activity was tested by the addition of a metacaspase specific peptide that is conjugated to the fluorescent reporter molecule. The cleavage of the peptide by the metacaspase releases the fluorochrome that, when excited by light, emits fluorescence. The level of metacaspase enzymatic activity in the cell lysate is directly proportional to the fluorescence signal detected. The use of specific standards in this test enables the quantification of the fluorescence results. This assay directly allows monitoring the metacaspase cleavage products and thereby tracing evidence for programmed cell death.