Assay of calpains and calpastatin
Calpain activity was determined using casein as a
substrate. Aliquots (1ml) of the column fractions
were incubated at 25C with 1ml of 0.7% casein in 100
mM Tris-HCl, 10 mM mercaptoethanol, 1 mM NaN3,
pH 7.5 containing 100 ml of either 0.1 M CaCl2 or 0.2 M
EDTA. The reaction was stopped after 30 min with 2 ml
of 5% TCA and the mixture centrifuged at 3000 g for 15
min. The absorbance of the supernatant was read at 278
nm. One unit of calpain was de®ned as the amount
which gave a Ca-dependent increase of 1.0 unit of
absorption in 1 h. Calpastatin was determined and
expressed as inhibitory equivalents of m-calpain.
Following localisation of the fractions with active
calpains and calpastatin, the appropriate fractions were
pooled and the activity of the bulked fractions reassayed
accurately.
2.5. Analysis of data
Statistical analysis was by a Minitab version 8 computer
package. Curves were ®tted to the data using the
SigmaPlot computer package.