Retinas were removed from cultures at PN3, immediately frozen at−
20°C, and analyzed for DEVD(Asp-Glu-Val-Asp)ase (DEVDase)
activity within 1 week. The frozen retinas were placed in 400 μl of a
room temperature buffer (50 mM Tris-HCl [pH 7.3 ], containing 100 mM
NaCl, 5 mM EDTA, 1 mM EGTA, 0.2% 3[3-cholaminopropyl
diethylammonio]-1-propane sulfonate [CHAPS], 3 mM NaN3,
1 mM phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin, 2.5 μg/ml
leupeptin, and 10 μg/ml aprotinin), vortexed, incubated in the
absence or presence of 50 μM z-Val-Ala-DL-Asp-fluoromethylketone
(zVAD-fmk; Bachem, Bubendorf, Switzerland) at 37°C for 45 minutes,
and centrifuged in a microfuge for 5 minutes. Samples of supernatants
(50 μl) were mixed with 50 μl of 50 μM
DEVD-7-amino-4-methylcoumarin (DEVD-AMC) substrate (Bachem) in the same
buffer (without CHAPS), and cleavage of DEVD-AMC was measured at room
temperature using a luminescence spectrometer (model LS 50B;
Perkin–Elmer, Norwalk, CT) with an excitation wavelength of 380 nm
(slit 10 nm) and emission wavelength of 460 nm (slit 15 nm). DEVD-AMC
cleavage was linear for 2 hours, and recovery of AMC was more than
95%. Fluorescence readings were obtained at 60 minutes and compared
with a standard curve of AMC in the same buffer. Protein was measured
by using the BCA protein assay (Pierce, Rockford, IL) using BSA as the
protein standard. The values presented represent data from three
retinas per treatment condition and are expressed as picomoles AMC
formed per minute per milligram protein.