Dynamic changes in the distribution of the calcium-activated neutral protease in human red blood cells following cellular insult and altered Ca2+ homeostasis.
Dynamic changes in the distribution of the calcium-activated neutral protease in human red blood cells following cellular insult and altered Ca2+ homeostasis.
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细胞损伤和 Ca2 稳态改变后，人红细胞中钙激活中性蛋白酶分布的动态变化。
DOI：
10.1016/0041-008x(92)90235-k
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发表时间：
1992
影响因子：
3.8
通讯作者：
Novak,RF
中科院分区：
文献类型：
--
作者：
Mortensen,AM;Novak,RF
我们进行了机制研究，以检验氧化膜蛋白损伤、Ca2+稳态改变和质膜结合Ca2+活化中性蛋白酶μCANP水平变化之间的关系。采用SDS-PAGE和免疫印迹法检测双氧水异丙苯（CHP）损伤后红细胞和溶血液中质膜结合μCANP水平的变化。研究了自由基清除剂、抗氧化剂和EGTA对chp处理细胞和溶血液中膜结合μCANP水平的影响。向红细胞中添加CHP （2mm）导致SDS-PAGE模式中许多蛋白带的强度显著降低/损失，包括带1、2、2.1、4.1、4.2和一个~ 60 kda的蛋白。n -乙酰半胱氨酸（20 mm）、二硫苏糖醇（50 mm）和二甲基硫脲（50 mm）可减轻chp介导的膜蛋白损伤；相反，二甲基呋喃（50 mm）加重了chp介导的膜蛋白损伤。二甲基亚砜（50 mm）无显著效果。自由基清除剂和抗氧化剂对膜结合μCANP水平的影响与它们调节膜蛋白损伤的能力在很大程度上是平行的。1 mm chp处理红细胞的免疫印迹分析显示膜结合μCANP的时间依赖性损失，在8小时监测μCANP的完全损失。用CHP处理红细胞也导致膜结合μCANP水平的浓度依赖性改变：在0.5或1.0 mm CHP时，检测到膜结合μCANP水平相对于对照降低，而在2至4 mm CHP时监测到结合酶水平升高。溶血液中添加CHP使膜结合μCANP水平降低，与红细胞中观察到的结果相当；在溶血液中加入Ca2+螯合剂EGTA或钙蛋白酶抑制剂I （n -乙酰-亮氨酸-亮氨酸-亮氨酸-亮氨酸-非亮氨酸）有效地抑制了这种减少。相比之下，在Ca2+离子载体A23187存在的情况下，用Ca2+处理红细胞导致SDS-PAGE蛋白带和膜结合μCANP水平的变化，与CHP产生的变化相当。EGTA的加入抑制了μCANP的结合。这些数据为氧化损伤后红细胞或溶血液中膜结合μCANP水平的动态变化提供了证据，并表明这一过程可以被自由基清除剂和抗氧化剂调节，通过在离子载体存在的情况下用Ca2+处理细胞来模拟，并被EGTA或Calpain Inhibitor I抑制。
Mechanistic studies were conducted to examine the relationship between oxidative membrane protein damage, altered Ca2+homeostasis, and changes in the levels of plasma membrane-bound Ca2+-activated neutral protease, μCANP. Alterations in the levels of plasma membrane-bound μCANP in erythrocytes and hemolysate following cumene hydroperoxide (CHP) insult were monitored using SDS-PAGE and immunoblot analyses. Free radical scavengers, antioxidant and EGTA effects on membrane-bound μCANP levels in CHP-treated cells and hemolysate were also examined. CHP (2 mm) addition to red cells caused a significant decrease/loss in intensity of numerous protein bands in the SDS-PAGE pattern, to include bands 1,2,2.1,4.1,4.2, and an ∼60-kDa protein. N-acetylcysteine (20 mm), dithiothreitol (50 mm), and dimethylthiourea (50 mm) diminished CHP-mediated membrane protein damage; in contrast, dimethylfuran (50 mm) exacerbated CHP-mediated membrane protein damage. Dimethylsulfoxide (50 mm) was without significant effect. The free radical scavengers and antioxidants differentially affected membrane-bound μCANP levels largely in parallel with their ability to modulate membrane protein damage. Immunoblot analysis of 1 mm CHP-treated red cells revealed a time-dependent loss of membrane-bound μCANP, with a complete loss of μCANP monitored at 8 hr. Treatment of erythrocytes with CHP also resulted in concentration-dependent alterations in the level of membrane-bound μCANP: at 0.5 or 1.0 mm CHP a decreased level of membrane-bound μCANP was detected relative to control, whereas an increase in the level of bound enzyme was monitored from 2 to 4 mm CHP. CHP addition to hemolysate produced a decrease in membrane-bound μCANP levels comparable to that observed with erythrocytes; addition of the Ca2+chelator EGTA or Calpain Inhibitor I (N-acetyl-leucyl-leucyl-leucyl-nor-leucinal) to hemolysate effectively inhibited this decrease. In contrast, treatment of erythrocytes with Ca2+in the presence of the Ca2+ionophore A23187 resulted in changes in the SDS-PAGE protein bands and membrane-bound μCANP levels that were comparable to those produced by CHP. Inclusion of EGTA in this system prevented μCANP binding. These data provide evidence for membrane damage and concomitant dynamic alterations in membrane-bound μCANP levels in the red cell or hemolysate following oxidative insult, and show that this process can be modulated by free radical scavengers and antioxidant, simulated by treating cells with Ca2+in the presence of ionophore, and inhibited by EGTA or Calpain Inhibitor I.
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DOI：
10.1083/jcb.85.2.361
发表时间：
1980-05
期刊：
The Journal of cell biology
影响因子：
--
作者：
Fowler V;Taylor DL
通讯作者：
Taylor DL
叔丁基过氧化氢诱导的人红细胞扰动作为氧化应激的模型。
DOI：
10.1016/0005-2736(85)90370-0
发表时间：
1985
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Rice-Evans,C;Baysal,E;Pashby,DP;Hochstein,P
通讯作者：
Hochstein,P
从人红细胞中纯化 Ca2 刺激的 ATP 酶激活剂。
影响因子：
4.8
作者：
H. Jarrett;J. T. Penniston
通讯作者：
J. T. Penniston
影响因子：
56.9
作者：
JEWELL, SA;BELLOMO, G;SMITH, MT
通讯作者：
SMITH, MT
人红细胞受到苯肼损伤后，蛋白水解作用增强，膜相关钙蛋白酶发生变化。
影响因子：
3.8
作者：
Mortensen,AM;Novak,RF
通讯作者：
Novak,RF