Bellavite P, Papini E, Zeni L, Della Bianca V, Rossi F
Istituto di Patologia Generale dell'Università di Verona, Italy.
Free Radic Res Commun. 1985;1(1):11-29. doi: 10.3109/10715768509056533.
Highly active superoxide (O2-)-forming NADPH oxidase was extracted from plasmamembranes of phorbol-12-myristate-13-acetate-activated pig neutrophils and was partially purified by gel filtration chromatography. Oxidase activity copurified with cytochrome b-245 in an aggregate containing phospholipids and was almost completely separated from FAD and NAD(P)H-cytochrome c reductase. A polypeptide with molecular weight of 31,500 strictly paralleled the purification of NADPH oxidase, suggesting that it is a major component of the enzyme. The enzyme complex was then dissociated by high detergent and salt concentration and cytochrome b-245 was isolated by a further gel filtration chromatography, with a 147 fold purification with respect to the initial preparation. The cytochrome b-245 showed a 31,500 molecular weight by SDS electrophoresis, indicating that it is actually the component previously identified in the partially purified enzyme. The 31,500 protein was phosphorylated in enzyme preparations from activated but not from resting neutrophils, suggesting that phosphorylation of cytochrome b-245 is involved in the activation mechanism of the O2(-) -forming enzyme responsible for the respiratory burst in phagocytes.
从佛波醇-12-肉豆蔻酸酯-13-乙酸酯激活的猪中性粒细胞的质膜中提取出高活性的产超氧阴离子（O2-）的NADPH氧化酶，并通过凝胶过滤色谱法进行部分纯化。氧化酶活性与细胞色素b-245在一个含有磷脂的聚集体中共纯化，并且几乎与FAD和NAD（P）H-细胞色素c还原酶完全分离。一种分子量为31,500的多肽与NADPH氧化酶的纯化过程严格平行，表明它是该酶的主要成分。然后通过高浓度去污剂和盐使酶复合物解离，并通过进一步的凝胶过滤色谱法分离出细胞色素b-245，相对于初始制剂纯化了147倍。通过SDS电泳，细胞色素b-245显示出31,500的分子量，表明它实际上是先前在部分纯化的酶中鉴定出的成分。在来自活化但非静息中性粒细胞的酶制剂中，31,500蛋白发生了磷酸化，这表明细胞色素b-245的磷酸化参与了吞噬细胞中负责呼吸爆发的产O2（-）酶的激活机制。
