Reconstitution of neutrophil NADPH oxidase activity in the cell-free system by four components: p67-phox, p47-phox, p21rac1, and cytochrome b-245

• Published in: The Journal of biological chemistry Vol. 267; no. 24; pp. 16767 - 16770
• Main Authors: ABO, A, BOYHAN, A, WEST, I, THRASHER, A. J, SEGAL, A. W
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 25.08.1992
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Cell Membrane - enzymology; Cell-Free System; Cloning, Molecular; Cytochrome b Group - blood; Cytosol - enzymology; Enzymes and enzyme inhibitors; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Humans; Kinetics; NADH, NADPH Oxidoreductases - blood; NADH, NADPH Oxidoreductases - genetics; NADPH Oxidases; Neutrophils - enzymology; Oxidoreductases; Plasmids; Recombinant Proteins - metabolism; Human; Cytochrome b; Enzymatic activity; Enzyme; Reconstituted system; Neutrophil; Cell free system
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(18)41846-7

Activation of the NADPH oxidase of phagocytes in the cell-free system requires the association of several cytosolic components with membrane-bound cytochrome b. In this study we were able to fully reconstitute NADPH oxidase activity in the cell-free system with three recombinant proteins: p67-phox, p47-phox, p21rac1, and pure cytochrome b-245. Activity was dependent upon the concentration of the proteins, with maximal activity observed with roughly equimolar ratios of the cytochrome b and p67-phox (133 and 163 mol/s/mol, respectively) and concentrations of the other two proteins approximately 1 order of magnitude greater. No activity was observed in the absence of any one of these components. In addition, activation was dependent upon p21rac1 being preloaded with GTP, the cytochrome b being reconstituted with lipid, and the presence of FAD during activation. Half-maximal activity was observed at a concentration of NADPH of approximately 50 microM. These findings confirm our recent description of the membrane-bound cytochrome b as a FAD-containing flavocytochrome b containing the NADPH binding site, and implicate the three cytosolic proteins in its activation.