A microtechnique for neutrophil respiratory burst oxidase in a cell-free system--characterization of oxidase activation system.
1. A microtechnique for quantitating human neutrophil NADPH oxidase in a cell-free system is described. 2. This spectrophotometric discontinuous (fixed time) method is less material-consuming than existing methods and is more useful for experiments in which superoxide production by neutrophils must be measured in a large number of samples. 3. Measurement of NADPH oxidase using the new method can be accomplished in a final vol of 0.15 ml. 4. In the assay, neutrophil membranes solubilized with deoxycholate were incubated for 3 min with cytosolic fractions, magnesium, sodium dodecyl sulfate, and cytochrome c in the absence of NADPH to preincubate the oxidase before the addition of the reducing agent. 5. The reaction was started by adding NADPH and 2 min later terminated by adding superoxide dismutase. 6. The apparent Km for NADPH obtained by the new method was almost the same as that by the authorized method (39.2 +/- 3.1 SD vs 36.8 +/- 1.6). Activation of neutrophil NADPH oxidase was characterized using the new assay method.
Umeki
S
S
Department of Medicine, Kawasaki Medical School, Okayama, Japan.
DeLisle
D M
DM
D002462:Cell Membrane / Q000201:enzymology; D002474:Cell-Free System; D004789:Enzyme Activation; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008832:Microchemistry; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000494:pharmacology; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D013053:Spectrophotometry; D013696:Temperature
D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases