NAD(P)H-dependent superoxide production by phagocytic vesicles from guinea pig and human granulocytes.

Superoxide production by granulocytes stimulated by surface active agents is the result of the activation of a plasma membrane-bound NAD(P)H oxidase. To determine whether this enzyme was also responsible for superoxide production by particle-stimulated cells, we prepared phagocytic vesicles from guinea pig granulocytes. These vesicles produce 34.5 f 1.8 mol of superoxide/min/mg of protein in the presence of NADPH (1 111~) compared with 13.0 2 0.6 nmol of superoxide/min/mg of protein for the cell homogenate from which they were prepared. Similar vesicle activities are obtained using 2 m~ NADH (27.8 f 2.0) or both NADPH and NADH (38.2 f 3.3) as substrate. This activity is present in vesicles made from human granulocytes and absent from vesicles made from granulocytes of patients with chronic granulomatous disease. The apparent K, for NADPH and NADH, the pH optimum, and the effects of 2-deoxyglucose, cyanide, and divalent cations are similar for the vesicle activity and that previously found for the plasma membrane enzyme. NADPH-dependent superoxide production in homogenates of particle-treated cells is maximal after 1 to 2 min, whereas maximal vesicle activity does not occur for 4 to 7 min. Continued incubation of cells with particles results in a loss of over 90% of the superoxidegenerating activity in the vesicles but only 65% in the homogenate. This inactivation occurs despite the retention of vesicle 5'-nucleotidase, P-glucuronidase, lysozyme, and total protein. We conclude that the same superoxide-generating enzyme is present in the plasma membrane and phagocytic vesicle, that activation of this enzyme precedes formation of the vesicle, and that inactivation occurs more rapidly in the phagocytic vesicle than in the whole cell.