McGinnis K M, Gnegy M E, Park Y H, Mukerjee N, Wang K K
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
Biochem Biophys Res Commun. 1999 Sep 16;263(1):94-9. doi: 10.1006/bbrc.1999.1315.
We demonstrate here that both procaspase-3 (32 kDa) and PARP are calpain substrates. In calcium-channel opener maitotoxin-treated cells, a 30 kDa caspase-3 fragment is produced in a time and concentration-dependent manner. Formation of this fragment is prevented by calpain inhibitors but not by the pancaspase inhibitor, carbobenzoxy-Asp-CH(2)OC(O)-2,6-dichlorobenzene (Z-D-DCB) nor the selective proteasome inhibitor lactacystin. In maitotoxin-treated cells, PARP (113 kDa) is also cleaved into a 40 kDa immunoreactive fragment, in a calpain-inhibitor-sensitive manner. Both procaspase-3 and PARP are also cleaved in vitro by purified micro-calpain to a 30 kDa fragment and a 40 kDa fragment, respectively. Finally, we show that staurosporine-mediated caspase-3 activation is interrupted by maitotoxin pretreatment.
我们在此证明，前体半胱天冬酶-3（32 kDa）和聚（ADP-核糖）聚合酶（PARP）都是钙蛋白酶的底物。在经钙通道开放剂 maitotoxin 处理的细胞中，会以时间和浓度依赖的方式产生一个 30 kDa 的半胱天冬酶-3 片段。该片段的形成可被钙蛋白酶抑制剂阻止，但不能被泛半胱天冬酶抑制剂苄氧羰基-天冬氨酸-CH(2)OC(O)-2,6-二氯苯（Z-D-DCB）或选择性蛋白酶体抑制剂乳胞素阻止。在经 maitotoxin 处理的细胞中，PARP（113 kDa）也会以对钙蛋白酶抑制剂敏感的方式被切割成一个 40 kDa 的免疫反应性片段。前体半胱天冬酶-3 和 PARP 在体外也分别被纯化的微钙蛋白酶切割成 30 kDa 的片段和 40 kDa 的片段。最后，我们表明，毒胡萝卜素介导的半胱天冬酶-3 激活会被 maitotoxin 预处理中断。
