NADPH oxidase from polymorphonuclear cells

This chapter presents nicotinamide adenine dinucleotide phosphate (NADPH) oxidase from polymorphonuclear cells. The NADPH oxidase enzyme system from polymorphonuclear leukocytes is responsible for the sudden burst of oxygen consumption that follows the exposure of these cells to certain activating agents. The enzyme system catalyzes the univalent reduction of molecular oxygen at the expense of pyridine nucleotide (NADPH) oxidation. The membrane-associated enzyme system has a vectorial orientation in the plasma membrane or phagosomal membrane of the cell. Oxidase activation is a highly specific process controlled by complex mechanisms at the cellular level. Polymorphonuclear leukocytes are prepared from fresh whole blood anticoagulated with acid-citrate-dextrose by dextran sedimentation followed by centrifugation over lymphoprep and hypotonic lysis of red blood cells. Cell suspension is disrupted by sonication for three 15 seconds intervals at 20 W power with the standard probe tip of a Branson 200 sonifier set on pulsed power, 50% duty cycle. For different sonifiers, the power and time of sonification should be adjusted so that only 70–80% of the cells are completely disrupted.