Escriou V, Laporte F, Vignais P V
Laboratoire de Biochimie (URA 1130/CNRS), Département de Biologie Moléculaire et Structurale, Grenoble, France.
Biochem Biophys Res Commun. 1996 Feb 27;219(3):930-5. doi: 10.1006/bbrc.1996.0335.
The latent NADPH oxidase activity of purified cytochrome b(558) from rabbit peritoneal neutrophils was expressed in a cell-free system consisting of either gel-filtrated cytosol from resting neutrophils, or a mixture of the three cytosolic activation factors, namely p47, p67 and the G protein Rac1. The cell-free system was supplemented with arachidonic acid and GTPgammaS. With gel-filtrated cytosol, the oxidase activity was relatively high (22 moles O(2)(-)/s/mole heme b in the absence of added FAD), and enhanced by less than one fourth upon addition of FAD. In contrast, with the purified cytosolic activation factors the rate of O(2)(-) production was low (8 moles O(2)(-)/s/mole heme b), and enhanced more than two-fold by a saturating concentration of FAD. The specificity of FAD was demonstrated by the lack of effect of FMN. FAD was determined together with heme b and the oxidase activity in eluates from a Sepharcryl column at the last step of the purification of cytochrome b(558). In the eluted fraction that contained both the maximal inducible oxidase activity and the highest amount of heme b, the molar amount of FAD was 20 times less than that of heme b. It is concluded that cytochrome b(558) is an NADPH-dependent flavocytochrome oxido-reductase (NADPH oxidase) in which one part of FAD is firmly bound and another, loosely attached. On the other hand, there may exist a parallel pathway of electron transfer from NADPH via distinct FAD dehydrogenase(s) to the heme b component of the NADPH oxidase.
从兔腹膜中性粒细胞中纯化得到的细胞色素b(558)的潜在NADPH氧化酶活性，在由静息中性粒细胞经凝胶过滤得到的胞质溶胶，或三种胞质激活因子（即p47、p67和G蛋白Rac1）的混合物组成的无细胞体系中得以表达。该无细胞体系添加了花生四烯酸和GTPγS。对于经凝胶过滤的胞质溶胶，氧化酶活性相对较高（在未添加FAD时为22摩尔O(2)(-)/秒/摩尔血红素b），添加FAD后活性增强不到四分之一。相比之下，使用纯化的胞质激活因子时，O(2)(-)生成速率较低（8摩尔O(2)(-)/秒/摩尔血红素b），饱和浓度的FAD可使其活性增强两倍以上。FMN无作用证明了FAD的特异性。在细胞色素b(558)纯化的最后一步，从Sepharcryl柱洗脱液中测定FAD、血红素b和氧化酶活性。在含有最大诱导氧化酶活性和最高量血红素b的洗脱组分中，FAD的摩尔量比血红素b少20倍。结论是细胞色素b(558)是一种NADPH依赖性黄素细胞色素氧化还原酶（NADPH氧化酶），其中一部分FAD紧密结合，另一部分松散结合。另一方面，可能存在一条从NADPH经不同的FAD脱氢酶到NADPH氧化酶血红素b组分的平行电子传递途径。
