Quantitative measurement of caspase-3 activity in a living starfish egg.
If not fertilized, synchronous apoptosis is induced in starfish eggs at approximately 11h after stimulation with the hormone, 1-methyladenine. In this study, a membrane-impermeant substrate of caspase-3, acetyl-Asp-Glu-Val-Asp-coumarylamido-4-methanesulfonic acid (Ac-DEVD-CAMS), was synthesized and microinjected into a starfish egg. Caspase-3 activity in unfertilized egg was detected approximately 30min before blebbing by quantifying the accumulation rate of a membrane-impermeant, fluorogenic product, 7-aminocoumarin-4-methanesulfonic acid (ACMS), using a photomultiplier mounted on a fluorescence microscope. When active recombinant human caspase-3 was microinjected into an egg at 3h after 1-methyladenine treatment, the injected caspase-3 activity decreased and disappeared within 2h. This decrease is probably due to proteasome-dependent degradation of the enzyme, since the injected caspase-3 was degraded and a proteasome inhibitor blocked its degradation. In contrast, in aged eggs at approximately 10h after 1-methyladenine treatment, no degradation of the injected caspase-3 was observed, suggesting that endogenous caspase-3 may stabilize at this point, therefore, inducing apoptosis.
Sakaue
Miki
M
Department of Biology, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo, Tokyo 112-8610, Japan.
Motoyama
Yumiko
Y
Yamamoto
Kayono
K
Shiba
Tetsuo
T
Teshima
Tadashi
T
Chiba
Kazuyoshi
K
D000818:Animals; D053148:Caspase 3 / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation; D009865:Oocytes / Q000201:enzymology*; D010063:Ovum / Q000201:enzymology*; D013215:Starfish / Q000196:embryology* / Q000201:enzymology*
D053148:Caspase 3