Abstract
Pig blood neutrophils were briefly activated by various fatty acids and then fractionated into membrane vesicles with different NADPH oxidase activities. Treatment of these membranes with a detergent, octyl glucoside, resulted in a high yield of solubilized oxidase, which was subjected to isoelectric focusing on gels (pI 4.0-8.0). 1) A distinct band staining with NADPH-nitroblue tetrazolium focused at pI 5.0. The enzyme (pI 5.0) showed high specificity for NADPH and similar characteristics to the oxidase involved in the respiratory burst. 2) The enzyme was extracted from gel slices and analyzed. When measured promptly after its extraction, its NADPH oxidase activity was high, but there was apparent superoxide dismutase-insensitive cytochrome c reduction, probably due to direct electron transfer to the heme protein. However, it could produce superoxide anion (O2-) under some micelle conditions. 3) Therefore, the formation of the enzyme-substrate complex of yeast cytochrome c peroxidase was employed for the detection of H2O2. A fresh extract of stimulated cells catalyzed equimolar NADPH oxidation and H2O2 production of 306 and 300 nmol min-1 (mg protein)-1, respectively. The Km value of the enzyme for NADPH was 30 +/- 13 (S.D.) microM. The recovery of the extract (pI 5.0) was 19% of the total activity. 4) The enzyme extract contained 1.1-1.9 nmol of FAD/mg of protein, giving a turnover number of 300-600 min-1 in terms of O2- generation/FAD. No heme protein was found in the enzyme. The enzyme was mainly of 67-kDa molecular mass.
Highlights
Katsuko Kakinuma$, Yukiko Fukuhara, and Mizuho Kaneda From the TokyoMetropolitun Institute of Medical ScienctE, Honkomagome 3-18-22, Bunkyo-ku, Tokyo 113, Japan
The reduction of NBT by neutrophils is associated with the respiratory burst (30)
On isoelectric focusing (IEF) of the oxidase solubilized with octyl glucoside, a distinct band of an NADPH oxidoreductase activity focused at PI 5.0 on the gel
Summary
When measured promptly after its extraction, its NADPH oxidase activity was high, but there was apparent superoxide dismutaseinsensitive cytochrome c reduction, probably due to direct electron transfer to the heme protein. It couldproduce superoxide anion (0;) under some micelle conditions.3)the formation of the nents as its redox center(s): a flavoprotein (8-11), a b-type cytochrome (12, 13), and ubiquinone (14, 15).The participation of a redox center(s) in theoxidase system is still controversial, but thefollowing evidence suggestthsat a flavoprotein is probably a component of the oxidase. The K,,v,alue that probably participates in the NADPH oxidase system as of the enzyme for NADPH was 30 2 13 (S.D.) WM.The an electron-transferring redox center. recovery of the extract (PI 5.0) was 19% of the total activity. 4) The enzyme extract contained 1.1-1.9 nmol of FAD/mg ofprotein, giving a turnover number
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
