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This approach is closer to in vivo assay and allows tracking activation
or suppression of metacaspase activity in different tissues or at differ-
ent developmental stages or treatments. One should keep in mind,
however, that most organisms contain more than one metacaspase
gene and all metacaspases present in the cell lysate will contribute to
the detected activity due to low stringency of metacaspase substrate-
specifi city. The assay can be optimized by using samples where a
single metacaspase is expressed or by using multiple metacaspase
knockout or knockdown organisms. We have optimized conditions
for measuring metacaspase activity in the cell lysates from Norway
spruce embryogenic cell cultures (Fig. 1 ).- Grind plant material in liquid nitrogen.
- Mix 100 mg of the ground material with 100 μL of the reac-
tion buffer without calcium and vortex. - Centrifuge the samples at 17,000 × g for 15 min at 4 °C.
- Transfer the supernatant into new Eppendorf tube. If required
fi lter it through four layers of miracloth. Keep samples cold
on ice.
3.5 Measurement
of Metacaspase
Activity in Cell Lysates
Fig. 1 Cleavage of EGR-AMC in cell lysates prepared from Norway spruce
embryogenic culture. Withdrawal of plant growth regulators (PGR), auxin and
cytokinin, induces development of early embryos and developmental cell death
accompanied by increase in mcII-Pa activity. Cultures were incubated for 5 days
either in proliferation medium containing PGR (+PGR) or in embryogenesis
inducing medium lacking PGR (−PGR). The cell lysates were prepared with and
without 50 mM CaCl 2. Proteolytic activity was measured by detecting increase of
AMC fl uorescence. ( a ) Kinetics of fl uorescence increase over time. Solid line and
dashed line curves represent reactions with and without CaCl 2 , respectively. Gray
and black curves represent reactions containing lysates from cells growing with
and without PGR, respectively. ( b ) The kinetics curves shown on panel ( a ) and
standard curve were used to convert relative fl uorescence units into picomoles
of free AMC released in one min per milligram of total proteinElena A. Minina et al.http://www.ebook3000.com