Enhanced proteolysis and changes in membrane-associated calpain following phenylhydrazine insult to human red cells.
Enhanced proteolysis and changes in membrane-associated calpain following phenylhydrazine insult to human red cells.
复制标题
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。
DOI：
10.1016/0041-008x(91)90045-g
复制
发表时间：
1991
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
采用高效液相色谱、一维和二维十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法(SDS-PAGE)以及主要膜蛋白的免疫印迹分析，研究了苯肼对人红细胞蛋白质的损伤作用。免疫印迹分析也检测了联氨损伤后钙激活的中性蛋白水解酶--钙蛋白酶与膜蛋白的关系。用高效液相色谱法测定红细胞悬液中的氨基酸含量，以定量测定蛋白质的降解。苯肼(4 MM)使亮氨酸、赖氨酸和组氨酸的释放率分别增加12倍、7倍和5倍∼。N-乙酰半胱氨酸(20 MM)、二硫苏糖醇(50 MM)和二甲基硫脲(50 MM)可使苯肼刺激的氨基酸释放率降低30-50%，而自由基清除剂和抗氧化剂二甲基呋喃(50 MM)和二甲基亚砜(50 MM)对∼无明显影响。钙离子螯合剂EGTA(10 MM)可抑制苯肼刺激的蛋白水解率(∼为30%)。苯肼(4 MM)使∼-PAGE图谱中的主要膜蛋白条带减弱，并在DNA28 kDa区域出现广泛的涂抹。SDS-PAGE显示，自由基清除剂和抗氧化剂不能明显改善苯肼处理的细胞膜蛋白损伤。对血影蛋白的免疫印迹分析证实了这些结果。然而，与对照相比，经苯肼处理后的膜蛋白的二维SDS-PAGE显示出现了新的蛋白质斑点，并丢失了现有的蛋白质斑点。此外，还对膜相关钙蛋白(79 kDa(酶原)、77 kDa和75 kDa形式)进行了蛋白质印迹分析。与对照组相比，苯肼处理的红细胞显示出与膜相关的前痛水平的浓度和时间依赖的变化。在苯肼存在的情况下，N-乙酰半胱氨酸、二硫苏糖醇、二甲基硫脲和二甲基亚砜似乎能保持与膜蛋白相关的原疼痛水平，但只有N-乙酰半胱氨酸和二硫苏糖醇保护77和75 kDa的形式。相比之下，二甲基呋喃在苯肼的存在下，导致所有三种形式的膜相关钙蛋白酶都显著减少。在苯肼处理的溶血物中，77和75 kDa形式的膜相关钙蛋白酶的水平比对照降低。当加入EGTA(10 MM)孵育时，这些形式消失，酶原水平降低。这些数据表明，在肼损伤后，钙蛋白酶被募集到细胞膜上，经历了一种钙依赖的向活性形式的转换，并可能参与了受损胞浆和膜蛋白的降解(S)。
Phenylhydrazine-mediated protein damage in human red cells has been assessed using HPLC, one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblot analysis of major membrane proteins. The association of the Ca2+-activated neutral protease, calpain, with membrane proteins following hydrazine insult was also examined using immunoblot analysis. HPLC amino acid analysis of red cell suspensions was employed to quantify proteolysis. Phenylhydrazine (4 mm) increased the rate of leucine, lysine, and histidine release by ∼12-, 7-, and 5-fold, respectively. N-acetylcysteine (20 mm), dithiothreitol (50 mm), and dimethylthiourea (50 mm) decreased the rate of phenylhydrazine-stimulated amino acid release by ∼30–50%; in contrast, the free radical scavengers and antioxidants dimethylfuran (50 mm) and dimethyl sulfoxide (50 mm) were without significant effect. The calcium chelator, EGTA (10 mm), inhibited phenylhydrazine-stimulated proteolysis by ∼30%. Phenylhydrazine (4 mm) caused attenuation of the major membrane protein bands present in the SDS-PAGE pattern and extensive smearing of a band in the region of ∼28 kDa. Free radical scavengers and antioxidants failed to ameliorate significantly membrane protein damage in phenylhydrazine-treated cells as judged by SDS-PAGE. Immunoblot analysis of spectrin confirmed these results. Two-dimensional SDS-PAGE of membrane proteins following phenylhydrazine treatment, however, revealed the appearance of new protein spots and a loss of existing protein spots as compared to control. Western blot analysis of membrane-associated calpain (79 kDa (proenzyme), 77- and 75-kDa forms) was also performed. Phenylhydrazine-treated red blood cells exhibited concentration- and time-dependent changes in the level of membrane-associated procalpain relative to control. The inhibitors N-acetylcysteine, dithiothreitol, dimethylthiourea, and dimethyl sulfoxide in the presence of phenylhydrazine appeared to preserve the level of procalpain in association with the membrane proteins, but only N-acetylcysteine and dithiothreitol protected the 77- and 75-kDa forms. In contrast, dimethylfuran in the presence of phenylhydrazine caused a substantial decrease in all three forms of membrane-associated calpain. In phenylhydrazine-treated hemolysate, the level of the 77- and 75-kDa forms of membrane-associated calpain was decreased relative to control. These forms were absent when EGTA (10 mm) was included in the incubation and the level of proenzyme was decreased. These data suggest that calpain is recruited to the membrane following hydrazine insult, undergoes a Ca2+-dependent conversion to the active forms, and may be involved in the degradation of damaged cytosolic and membrane protein(s).