脱氢抗坏血未服用的净化、克隆和表达活动从人类中性粒细胞:glutaredoxin识别。
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公园JB,莱文M
脱氢抗坏血未服用的净化、克隆和表达活动从人类中性粒细胞:glutaredoxin识别。
j . 1996 5月1日,315 (Pt 3): 931 - 8。
- PubMed ID
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8645179 (在PubMed]
- 文摘
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脱氢抗坏血未服用活动正常的人类嗜中性粒细胞溶解产物特征,确定基于活动的净化和测量的新合成抗坏血酸盐通过高效液相色谱法。最初的减少活动non-dialysable和不能占作为还原剂谷胱甘肽的活性。减少活动是同质性作为11 kDa蛋白质纯化。蛋白质的特定活动3 mumol /分钟/毫克的蛋白质和依赖谷胱甘肽。动力学实验表明,谷胱甘肽的蛋白质有K (m)的2.0毫米和K (m) 250 microM脱氢抗坏血酸。脱氢抗坏血酸还原纯化蛋白依赖pH值和最大pH值7.5。肽片段纯化蛋白氨基酸序列进行分析和蛋白质被确认为glutaredoxin。利用简并基于氨基酸序列的寡核苷酸,glutaredoxin从图书馆克隆人类嗜中性粒细胞。表达纯化glutaredoxin显示减少活动和动力学与本地的纯化酶是没有区别的。几种方法表明glutaredoxin负责大部分的protein-mediated脱氢抗坏血酸降低溶解产物。 From protein purification data, glutaredoxin was responsible for at least 47% of the initial reducing activity. Dehydroascorbic acid reduction was at least 5-fold greater in neutrophil lysates than in myeloid tumour cell lysates, and glutaredoxin was detected in normal neutrophil lysates but not in myeloid tumour cell lysates by Western blotting. Glutaredoxin inhibitors inhibited dehydroascorbic acid reduction in neutrophil lysates as much as 80%. These findings indicate that glutaredoxin plays a major role in dehydroascorbic acid reduction in normal human neutrophil lysates, and represent the first identification of dehydroascorbic acid reductase in human tissue by activity-based purification.