Koshkin V, Pick E
Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Israel.
FEBS Lett. 1994 Feb 7;338(3):285-9. doi: 10.1016/0014-5793(94)80285-8.
Purified cytochrome b559 relipidated with either a mixture of phosphatidylcholine and phosphatidic acid or with phosphatidylcholine only exhibits high and low superoxide (O2-) producing ability, respectively, in the absence of cytosolic activators [Koshkin, V. and Pick, E. (1993) FEBS Lett. 327, 57-62]. This system was used as a model for the study of the mechanism of NADPH oxidase activation. It is shown that, depending on the composition of the phospholipid environment, cytochrome b599 binds FAD with high or low affinity, this being accompanied by changes in flavin absorbance and fluorescence. High affinity binding of FAD to cytochrome b559 relipidated with phosphatidylcholine combined with phosphatidic acid is associated with an enhanced NADPH-driven O2- producing capacity. A kinetic study of O2- production by cytochrome b559 reflavinated under stoichiometric FAD binding conditions revealed an FAD/heme ratio of 1:2. A further kinetic study of O2- production by high- and low-activity relipidated and reflavinated cytochrome b559, at varying substrate concentrations, and the determination of steady-state difference spectra of such preparations, reduced by NADPH, indicated that O2- production is activated by facilitation of electron transfer from NADPH to FAD rather than by an enhancement of NADPH binding.
在没有胞质激活剂的情况下，用磷脂酰胆碱和磷脂酸混合物或仅用磷脂酰胆碱重新脂质化的纯化细胞色素b559分别表现出高和低的超氧化物（O2-）产生能力[科什金，V.和皮克，E.（1993年）《欧洲生物化学学会联合会快报》327，57 - 62]。该系统被用作研究NADPH氧化酶激活机制的模型。结果表明，根据磷脂环境的组成，细胞色素b599以高或低亲和力结合FAD，这伴随着黄素吸光度和荧光的变化。FAD与用磷脂酰胆碱和磷脂酸重新脂质化的细胞色素b559的高亲和力结合与增强的NADPH驱动的O2-产生能力相关。在化学计量的FAD结合条件下对重新黄素化的细胞色素b559产生O2-的动力学研究揭示了FAD/血红素比率为1:2。对高活性和低活性重新脂质化和重新黄素化的细胞色素b559在不同底物浓度下产生O2-的进一步动力学研究以及对用NADPH还原的此类制剂的稳态差光谱的测定表明，O2-的产生是通过促进电子从NADPH转移到FAD而不是通过增强NADPH结合来激活的。
