title: Stabilization of human neutrophil NADPH oxidase activated in a cell-free system by cytosolic proteins and by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.
abstract: The superoxide-generating respiratory burst oxidase (NADPH oxidase) from human neutrophils can be activated in a cell-free system consisting of plasma membrane and cytosol by anionic amphiphiles such as sodium dodecyl sulfate and arachidonate (McPhail, L. C., Shirley, P. S., Clayton, C. C., and Snyderman, R. (1985) J. Clin. Invest. 75, 1735-1739; Curnutte, J. T. (1985) J. Clin. Invest. 75, 1740-1743; Bromberg, Y., and Pick, E. (1984) Cell. Immunol. 88, 213-221). Herein, the activity thus obtained is shown to be very labile at 37 degrees C. The rate of inactivation varied inversely with cytosol concentration. The stabilizing factor(s) was destroyed by heat and trypsin, indicating that it is protein in nature. Whereas cytosol from normal cells and from a chronic granulomatous disease patient lacking p67phox stabilized the oxidase activity, that from a chronic granulomatous disease patient lacking p47phox did not. Also, dialdehyde NADPH-treated cytosol showed no stabilizing effect, indicating that p47phox and a putative NADPH-binding component both participate in stabilization. The mechanism of inactivation was further explored by examining the stabilizing effect of agents that can act as chemical cross-linkers. Of several tested, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was the most effective, but others that utilize different chemical mechanisms were also partially effective. EDC extended the half-life at 37 degrees C from 2 to 120 min, protected against the inactivating effects of Triton X-100 and high salt, and did not affect the Km for NADPH. Stabilization required prior activation in the presence of both cytosol and membrane; and EDC treatment of cytosol, membrane, or a mixture of the two prior to the addition of sodium dodecyl sulfate failed to induce stabilization. EDC eliminated the requirement for the continuous presence of cytosol and activator. Dialysis did not cause a loss in activity, whereas control activity was diminished with dialysis and was largely restored with added sodium dodecyl sulfate. In the absence of EDC, the separation of cytosol from the membrane fraction resulted in a significant loss of activity, which was largely restored by the addition of cytosol. However, EDC treatment allowed the isolation of a nearly fully active oxidase in the membrane fraction, the activity of which was not influenced by added cytosol. These results support a model in which the active NADPH oxidase consists of a dissociable complex among membrane and cytosolic components and indicate that the longevity of the activated state requires continuous association of these components.
authors.affiliation: Department of Biochemistry, Medical College of Oita, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Tamura
authors.forename: M
authors.initials: M
authors.lastname: Takeshita
authors.forename: J T
authors.initials: JT
authors.lastname: Curnutte
authors.forename: D J
authors.initials: DJ
authors.lastname: Uhlinger
authors.forename: J D
authors.initials: JD
authors.lastname: Lambeth
mesh_terms: D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002474:Cell-Free System; D003432:Cross-Linking Reagents; D003600:Cytosol / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology*; D006207:Half-Life; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D011506:Proteins / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D011506:Proteins; D013481:Superoxides; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D005022:Ethyldimethylaminopropyl Carbodiimide