Purification and some properties of the 45 kDa diphenylene iodonium-binding flavoprotein of neutrophil NADPH oxidase
| Autor: | Andrew R. Cross, Owen T.G. Jones, C M Yea |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 1990 |
| Předmět: |
Cytochrome
Neutrophils Fluorescence spectrometry Flavoprotein Biochemistry chemistry.chemical_compound Onium Compounds Affinity chromatography Superoxides Humans NADH NADPH Oxidoreductases Molecular Biology Dihydrolipoamide Dehydrogenase Oxidase test NADPH oxidase Dihydrolipoamide dehydrogenase biology Cell-Free System Flavoproteins Chemistry Superoxide NADPH Oxidases Cell Biology Molecular biology Spectrometry Fluorescence biology.protein Chromatography Gel Electrophoresis Polyacrylamide Gel Isoelectric Focusing Research Article |
| Popis: | The 45 kDa diphenylene iodonium-binding flavoprotein of the human neutrophil superoxide-generating oxidase has been purified by affinity chromatography. The polypeptide was eluted from Blue Memsep or 2′,5′-ADP-agarose columns with either NADP or low concentrations of the specific inhibitor diphenylene iodonium. The purified protein was shown to bind FAD at a ratio of 1.09 mol of FAD/mol of protein. The reconstituted flavoprotein had a fluorescence spectrum similar, but not identical, to that of free FAD. It had an isoelectric point of approx. 4.0. The reconstituted flavoprotein displayed no diaphorase activity towards a range of artificial electron acceptors. Polyclonal antibodies raised against the pure protein inhibited superoxide generation by solubilized oxidase in a dose-dependent manner, and inhibited superoxide generation when incubated with either cytosol or membrane fractions in a reconstituted system. These antibodies precipitated the 45 kDa polypeptide together with a haem-containing 23 kDa protein thought to be the small subunit of cytochrome b-245. Antibodies raised against cytochrome P-450 reductase also precipitated these two polypeptides. These results are consistent with the 45 kDa polypeptide being the flavoprotein of the neutrophil superoxide-generating oxidase. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|