Freeman J L, Lambeth J D
Department of Biochemistry, Emory University Medical School, Atlanta, Georgia 30322, USA.
J Biol Chem. 1996 Sep 13;271(37):22578-82. doi: 10.1074/jbc.271.37.22578.
The neutrophil superoxide generating NADPH oxidase is activated by the assembly of cytosolic protein components with a membrane-associated flavocytochrome. The activity can be reconstituted in vitro using purified cytosolic factors p47(phox), p67(phox), and Rac plus the phospholipid-reconstituted flavocytochrome b558. Here, we demonstrate that activity is reconstituted in the absence of p47(phox) when high concentrations of p67(phox) and Rac are used. Vmax values were the same in the presence or absence of p47(phox), yet p47(phox) increases the affinity of both p67(phox) and Rac for the oxidase complex by nearly 2 orders of magnitude. p67(phox)-(1-246), a truncated form of the protein which eliminates SH3 domains involved in binding to p47(phox), also supports superoxide generation, both in the presence and absence of p47(phox), providing further evidence for p47(phox) independent activity. In the absence of p47(phox), p67(phox)-(1-246) binds to the NADPH oxidase complex 3-fold more tightly than does native p67(phox), indicating that the C terminus contains a region which masks binding to the oxidase complex. Results indicate that p47(phox) does not play a direct role in regulating electron transfer. Rather, its function is to serve as an adaptor protein to enhance the assembly of the other cytosolic components with the flavocytochrome and possibly to unmask a binding region in the N terminus of p67(phox) by binding to its C-terminal domains. p67(phox) and/or Rac play a more direct role in regulating electron transfer.
中性粒细胞产生超氧化物的NADPH氧化酶通过胞质蛋白组分与膜相关黄素细胞色素的组装而被激活。该活性可在体外使用纯化的胞质因子p47（phox）、p67（phox）和Rac以及磷脂重构的黄素细胞色素b558进行重构。在此，我们证明当使用高浓度的p67（phox）和Rac时，在没有p47（phox）的情况下也能重构活性。无论有无p47（phox），Vmax值相同，但p47（phox）使p67（phox）和Rac对氧化酶复合物的亲和力增加了近2个数量级。p67（phox）-（1-246）是该蛋白的截短形式，消除了参与与p47（phox）结合的SH3结构域，无论有无p47（phox），它也都能支持超氧化物的产生，为不依赖p47（phox）的活性提供了进一步证据。在没有p47（phox）的情况下，p67（phox）-（1-246）与NADPH氧化酶复合物的结合比天然p67（phox）紧密3倍，表明C末端包含一个掩盖与氧化酶复合物结合的区域。结果表明，p47（phox）在调节电子传递中不发挥直接作用。相反，其功能是作为一种衔接蛋白，增强其他胞质组分与黄素细胞色素的组装，并可能通过与p67（phox）的C末端结构域结合来暴露p67（phox）N末端的一个结合区域。p67（phox）和/或Rac在调节电子传递中发挥更直接的作用。
