Further characterization of NADPH oxidase activity of human polymorphonuclear leukocytes.

Abstract

A B S T R A C T Mn2+ was shown to catalyze a nonenzymatic oxidation of NADPH in the presence of superoxide anion by means of an isotopic assay for measurement of the oxidation of NADPH to NADP+. Human polymorphonuclear leukocyte granule NADPH oxidase activity was evaluated in the absence of Mn2" and was found to be higher in granules from phagocytizing cells than in granules from resting cells. The drug phorbol myristate acetate, which affects the oxidative metabolism of the neutrophil like phagocytosis, was found to activate granule NADPH oxidase activity. Superoxide dismutase was shown to inhibit NADPH oxidase activity both in the presence and absence of added Mn2'. The NADPH oxidase reaction in the absence of Mn2" was optimal at pH 5.5, and was more linear with increasing time and protein concentration than in the presence of Mn2+. No activity was measurable in granules isolated from resting cells until the level of NADPH added was above 0.25 mM. Activity was present in granules isolated from cells challenged with opsonized zymosan, even at 0.05 mM NADPH, and was higher than the activity found in granule fractions from resting cells at all levels of NADPH tested. The addition of as little as 0.1 /AM NADH to the reaction mixture was found to inhibit granular NADPH oxidase activity, indicating a possible regulatory role for NADH. These results suggest that NADPH oxidase may be the enzyme that initiates the metabolic events accompanying phagocytosis.

Generation of chemiluminescence by a particulate fraction isolated from human neutrophils. Analysis of molecular events.

“…Peak of activity with A23187 was slightly delayed, occurring at 3-4 min. In parallel studies, A23187 also effectively stimulated NADPH oxidase activity in particulate fractions, as described previously for PMA and opsonized zymosan (13 A23187-treated, 1.05±0.11 (3) [mean±+SEM (n)].…”

“…(23)(24)(25)(26), either stimulated chemiluminescence or had no effect on activity. We examined the action of these agents on the chemiluminescence of the xanthine oxidase-acetaldehyde system, which is mediated by oxygen radicals (19 (13,31), which has been attributed to removal of the direct oxidative effects of O2 (31). Ethanol produced a 48% inhibition of NADPH-dependent°2 generation, thus its slight effect on chemiluminescence is difficult to interpret.…”

“…Previous attempts to identify the luminescing species have used certain scavengers and inhibitors; however, many ofthese compounds also affect other aspects of the phagocytic process in whole cells, such as binding and ingestion. We (12)(13)(14) and others (15)(16)(17) have shown NADPH-dependent oxidase activity and°2 production in a cell-free system isolated from human neutrophils. We now report that this system produces chemiluminescence that has characteristics like those of intact cells.…”

“…An equal volume of 0.68 M sucrose was added and samples were homogenized for 5 min (13). After a low speed (500 g) centrifugation, supernates were spun at 27,000 g (13).…”

Inhibition of neutrophil activation by p‐bromophenacyl bromide and its effects on phospholipase A2

“…In selected experiments the NADPH oxidase activity was determined in the 27,000 g particle fraction of sonicated neutrophils as previously described (McPhail et al, 1976). Briefly, rat neutrophils were divided into two aliquots.…”