Respiratory burst oxidase from human neutrophils: purification and some properties.

Abstract

The respiratory burst oxidase of human neutrophils was purified by "dye-affinity" chromatography over a red agarose column. Electrophoresis of the purified enzyme on NaDodSO4 gel showed a single major band at 64,000-66,000 daltons, together with some minor contaminants. On a nondenaturing gel, the enzyme ran as two closely spaced bands, the faster of which contained flavin. When these two bands were rerun separately on a NaDodSO4 gel, they gave identical patterns, each showing a major band at ca. 65,00Q daltons. The specific activity (mean ± SEM) of the purified enzyme was 8.8 ± 3.5 ,umol of O2 per min/mg of protein.When exposed to appropriate stimuli, neutrophils are induced to express the "respiratory burst," a profound alteration in oxygen metabolism whose purpose is the generation of microbicidal oxidants by the (12)(13)(14)(15)(16).The respiratory burst oxidase was first solubilized from the plasma membranes of activated human neutrophils in 1978 (17). We now report the purification of this oxidase and describe some of the properties of the purified enzyme. MATERIALS AND METHODSHuman neutrophils were prepared by dextran sedimentation and differential centrifugation as described (18). Zymosan (Sigma) was boiled for 10 min in 1 M NaOH, then washed three times with Hanks' balanced salt solution (GIBCO), and finally opsonized as described elsewhere (3). Superoxide dismutase, horse heart cytochrome c (type VI), phosphatidylethanolamine (ovine brain), Lubrol PX, sodium deoxycholate, phenylmethylsulfonyl fluoride, leupeptin, pepstatin, FAD, and NADPH were purchased from Sigma. The sodium deoxycholate was purified by recrystallization from absolute ethanol. Red Sepharose CL-6B (agarose coupled to reactive red 120) was obtained from Pharmacia. The concentration of dye in the lots purchased from Pharmacia ranged from 2.0 to 3.0 Afmol/ml of packed gel. Other reagents were the best grade commercially available apd were used without further purification.Gel Electrophoresis. Electrophoresis on 7.5% nondenaturing slab gels was performed by a modification of the method of Davis (19) in which the solution for preparing the lower gel contained 4% glycerol plus twice the usual concentration of Tris, that for preparing the upper gel contained half the usual concentration of bisacrylamide, and polymerization was accomplished with 0.035% (wt/vol) ammonium persulfate, which was removed by preelectrophoresis for 1 hr before the samples were applied to the slab. Nondenaturing gel electrophoresis was carried out at 40C. NaDodSO4/polyacrylamide gel electrophoresis (NaDodSO4/PAGE) was performed by the method of Laemmli (20) using 9o slab gels. Where necessary, samples were concentrated before electrophoresis in CF 25A ultrafiltration cones (Amicon) and then centrifuged for 30 min at 100,000 x g to remove aggregated proteins. Proteins were visualized with Coomassie blue R-250 or with a silver stain kit purchased from Bio-Rad.Localization of Flavin in the Nondenaturing Gel. The location of flavin in the nondenaturing gel was ...

Retinol Inhibition of in Vitro Human Neutrophil Superoxide Anion Release

“…NADPH oxidoreductase (0; synthetase) is known to be exquisitely sensitive to endogenous PMN proteases (29). Release of proteases from PMN granules by retinol could perhaps explain its inhibitory effect on 0; production.…”

The superoxide‐generating oxidase of phagocytic cells

“…A definite conclusion, however, had to await the purification of this flavoprotein and demonstration of its kinetic competency by reconstitution experiments. An NADPHbinding protein with a mass of 65-67 kDa, most likely a flavoprotein, has been purified from activated neutrophils isolated from human blood (Markert et al, 1985), bovine blood (Doussiere and Vignais, 1985) and pig blood (Kakinuma et al, 1987). This protein had an isoelectric point of 5 and a KM for NADPH of 30-40 pM, similar to that found for the oxidase complex in neutrophil membranes.…”

Two cytosolic components of the human neutrophil respiratory burst oxidase translocate to the plasma membrane during cell activation.

“…Both the 47-and 67-kD proteins have an affinity for 2',5'-ADP, a property suggestive of NADPH-binding capacity (23,42). A protein of 66-68 kD has been detected in a highly purified oxidase preparation from stimulated neutrophils (43,44), but its relationship to the cytosolic component of similar size is unknown. The use of the 2,3-dialdehyde derivative of NADPH as an affinity probe of the oxidase has identified a similar membrane component, but it is present in membranes of both resting and stimulated PMN (45, 46).…”