Cytosolic components involved in porcine neutrophil oxidase activation. Purification of a 47-kilodalton protein and reconstitution of the activation system.
Two cytosolic components, which cooperate with a 63-kDa cytosolic factor (Tanaka, T., Imajoh-Ohmi, S., Kanegasaki, S., Takagi, Y., Makino, R., and Ishimura, Y. (1990) J. Biol. Chem. 265, 18717-18720) in activation of the O(2-)-generating NADPH-oxidase in neutrophil membrane, were isolated and characterized from porcine neutrophils. One, which was purified to electrophoretic homogeneity, was a 47-kDa protein cross-reactive to an antibody raised against a portion of human 47-kDa cytosolic factor, a component of the human NADPH-oxidase activation system. Another one, designated here as the third component, was partially purified and found to contain 49- and 55-kDa proteins as the major constituents. No colored prosthetic group such as heme, flavin, and non-heme iron was detected in both cytosolic components. In a reconstituted assay system with a solubilized membrane preparation containing the dormant oxidase and with oleate as a stimulus, the 47-kDa protein together with the 63-kDa factor was essential for activating the dormant oxidase, while the third component was not essential for the activation of but enhanced the O(2-)-generation evoked by the former two components. Thus, the 47- and 63-kDa cytosolic proteins are the principal constituents of the activation system, while the other factors such as those contained in the third component may regulate the activity induced by the essential components.
Tanaka
T
T
Department of Biochemistry, Keio University, School of Medicine, Tokyo, Japan.
Makino
R
R
Ishimura
Y
Y
D000818:Animals; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D003600:Cytosol / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006801:Humans; D009247:NADH, NADPH Oxidoreductases / Q000097:blood* / Q000302:isolation & purification; D009504:Neutrophils / Q000201:enzymology*; D013481:Superoxides / Q000378:metabolism; D013552:Swine
D013481:Superoxides; D009247:NADH, NADPH Oxidoreductases