Stabilization of the superoxide-generating respiratory burst oxidase of human neutrophil plasma membrane by crosslinking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.
The superoxide-generating respiratory burst oxidase (NADPH-oxidase) of neutrophil plasma membranes is known to be highly unstable. In an attempt to stabilize the enzyme, we investigated the effect of crosslinking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The stability of superoxide-generating activity of plasma membrane was significantly enhanced by crosslinking. The half-life (t1/2) of the activity at 37 degrees C in the absence of crosslinker was about 2 min. Crosslinking extended the t1/2 significantly. Crosslinked material exhibited a biphasic loss of activity: about half was lost in each phase with respective t1/2 values of 20 and 240 min. The lifetime of the crosslinked material at 37 degrees C was further extended (about sixfold) with 30% glycerol, and the crosslinked material was completely stable for more than 2 weeks if stored on ice. Crosslinking also stabilized the activity to the effects of high salt and detergent, both of which have inactivating effects on the oxidase. In addition, crosslinking stabilizes not only the Vm but also the Km of the enzyme, which was noted to increase upon storage in the absence of crosslinking. Unlike the native material, the crosslinked oxidase failed to be stimulated (and in fact was inhibited) by phosphatidylserine, recently reported to be an activator of the oxidase (Tamura et al. (1988) J. Biol. Chem. 263, 17,621-17,626). The crosslinked plasma membrane provides a useful stabilized system for kinetic studies. When the activated plasma membrane was treated with EDC, the stabilized oxidase could not be solubilized effectively using detergents, since greater than 95% of the activity remained with the pellet following centrifugation, perhaps due to crosslinking to the cytoskeleton. However, when the activity was first detergent-solubilized, the soluble activity was also stabilized by EDC. This solubilized, crosslinked material may provide useful starting material for subsequent isolation and characterization of a stabilized active NADPH-oxidase.
Tamura
M
M
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322.
Tamura
T
T
Burnham
D N
DN
Uhlinger
D J
DJ
Lambeth
J D
JD
D002234:Carbodiimides / Q000494:pharmacology*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D004795:Enzyme Stability; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D011189:Potassium Chloride / Q000494:pharmacology; D013481:Superoxides / Q000097:blood*
D002234:Carbodiimides; D013481:Superoxides; D011189:Potassium Chloride; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C022709:superoxide-forming enzyme; D005022:Ethyldimethylaminopropyl Carbodiimide