Superoxide-forming NADPH oxidase preparation of pig polymorphonuclear leucocyte

Abstract

A phagocytic vesicle fraction with high NADPH-dependent superoxide-forming activity was obtained in large quantity from pig blood polymorphonuclear leucocytes, phagocytosing oil droplets in the presence of cyanide. The activity of the homogenate of the phagocytosing cells was 40 times that of the resting cells, and 70% of the activity in the homogenate was recovered in the phagocytic vesicle fraction. Essentially all of the superoxide-forming activity was extracted by repeated extraction with a mixture containing deoxycholate and Tween 20. The extract had a superoxide-forming activity of 1 mumol/min per mg of protein with NADPH, and one-fifth of this with NADH, Km values being similar to those of the vesicle fraction (40 microM for NADPH and 400 microM for NADH). A stoichiometric relationship of 1:2 for NADPH oxidation and superoxide formation was obtained, in agreement with the reaction NADPH +2O2 leads to NADP+ + 2O2 -. + H+. The activity of the extract was enhanced 2-fold by the addition of FAD, suggesting that the flavin is a component of the enzyme system. The Km value for FAD was 0.077 microM. The activities in both vesicle fraction and extract were labile even on refrigeration, but could be kept for several months at -70 degrees C.

Phagocyte NADPH Oxidase, but Not Inducible Nitric Oxide Synthase, Is Essential for Early Control of Burkholderia cepacia and Chromobacterium violaceum Infection in Mice

“…Among B. cepacia strains, a significant reduction in viable organisms occurred at 1 mM H 2 O 2 , a concentration that far exceeds the level of reactive oxidants produced in vesicular fractions of neutrophils (35). In vitro, both B. cepacia and C. C. violaceum BS 6.6 Ϯ 2. violaceum were sensitive to exogenous reactive nitrogen intermediates (Fig.…”

Chronic granulomatous disease with neutrophil membrane cytochrome b deficiency: Demonstration by immunochemical staining with monoclonal antibody.

“…A neutrophil-rich fraction was prepared from acid citrate-dextrose treated whole blood by polyvinyl-pyrrolidone sedimentation as previously reported (Wakayama et al 1982), followed by hypotonic lysis of residual red blood cells. The cell concentration of the TABLE 1.…”

The superoxide‐generating oxidase of phagocytic cells

“…Early studies, summarized below, lend support to the concept that this carrier is a flavoprotein with FAD as prosthetic group. (a) FAD was able to reactivate 0; production in particulate fractions of neutrophils treated with either Triton X-100 (Babior and Kipnes, 1977) or a mixture of deoxycholate and Tween 20 (Wakeyama et al, 1982). (b) 0; generation by extracts of activated neutrophils was inhibited by FAD analogs, which transfer electrons only in pairs, such as 5-deaza FAD (Light et al, 1981).…”