Studies on the nature and activation of O2(-)-forming NADPH oxidase of leukocytes. Identification of a phosphorylated component of the active enzyme.
Highly active superoxide (O2-)-forming NADPH oxidase was extracted from plasmamembranes of phorbol-12-myristate-13-acetate-activated pig neutrophils and was partially purified by gel filtration chromatography. Oxidase activity copurified with cytochrome b-245 in an aggregate containing phospholipids and was almost completely separated from FAD and NAD(P)H-cytochrome c reductase. A polypeptide with molecular weight of 31,500 strictly paralleled the purification of NADPH oxidase, suggesting that it is a major component of the enzyme. The enzyme complex was then dissociated by high detergent and salt concentration and cytochrome b-245 was isolated by a further gel filtration chromatography, with a 147 fold purification with respect to the initial preparation. The cytochrome b-245 showed a 31,500 molecular weight by SDS electrophoresis, indicating that it is actually the component previously identified in the partially purified enzyme. The 31,500 protein was phosphorylated in enzyme preparations from activated but not from resting neutrophils, suggesting that phosphorylation of cytochrome b-245 is involved in the activation mechanism of the O2(-) -forming enzyme responsible for the respiratory burst in phagocytes.
Bellavite
P
P
Istituto di Patologia Generale dell'Università di Verona, Italy.
Papini
E
E
Zeni
L
L
Della Bianca
V
V
Rossi
F
F
D000818:Animals; D002850:Chromatography, Gel; D003573:Cytochrome b Group / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010766:Phosphorylation; D013481:Superoxides / Q000378:metabolism*; D013552:Swine
D003573:Cytochrome b Group; C035130:cytochrome b245; D013481:Superoxides; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases