Superoxide production by cytochrome b 559

FEBS Letters

Pick, Edgar¹  Koshkin, Vasilij¹ 

[1] Laboratory of Immunopharmacology, Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel

关键词: Superoxide;    Cytochrome b 559;    NADPH oxidase;    Phospholipid;    FAD;    O2 −;    Superoxide;    SDS;    sodium dodecyl sulfate;    EGTA;    [ethylenebis(oxyethylenenitrilo)]tetracetic acid;    PMSF;    phenylmethyl-sulfonylfluoride;    SOD;    Superoxide dismutase;    LiDS;    lithium dodecyl sulfate;   

DOI  :  10.1016/0014-5793(94)80285-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Purified cytochrome b ₅₅₉ 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 b ₅₅₉ binds FAD with high or low affinity, this being accompanied by changes in flavin absorbance and fluorescence. High affinity binding of FAD to cytochrome b ₅₅₉ 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 b ₅₅₉ 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 eytochrome b ₅₅₉, 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.

【 授权许可】

Unknown