Purification of human neutrophil NADPH oxidase cytochrome b-558 and association with Rap 1A.
The availability of sufficient quantities of highly purified phagocyte cytochrome b-558 has been necessary for many of the biochemical and immunological analyses of this important NADPH oxidase component, and it was only through the analysis of highly purified cytochrome b that the subunit composition was elucidated and the small subunit (p22-phox) was cloned and sequenced. In addition, the association of the small GTP-binding protein Rap1A with cytochrome b-558 was discovered through the analysis of purified cytochrome b. The procedures described here provide an easy, efficient, and highly reproducible method for the purification of cytochrome b as well as cytochrome b-Rap1A complexes. The ability to purify cytochrome b and cytochrome b-Rap1A complexes will also allow further analysis of the structure of this novel plasma membrane redox protein and the role of its association with low molecular weight GTP-binding proteins in the structure and regulation of the phagocyte NADPH oxidase.
Montana State University, Bozeman 59717, USA.
M T
MT
Quinn
C A
CA
Parkos
A J
AJ
Jesaitis
D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D003573:Cytochrome b Group / Q000302:isolation & purification*; D019204:GTP-Binding Proteins / Q000302:isolation & purification*; D006801:Humans; D008954:Models, Biological; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D020668:rap GTP-Binding Proteins
D003573:Cytochrome b Group; C036888:cytochrome b558; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins