Comparison of NADH and NADPH oxidase activities in granules isolated from human polymorphonuclear leukocytes with a fluorometric assay.

Abstract

A B S T R A C T A fluorometric method for the determination of pyridine nucleotides has been adapted for use in studying the reduced pyridine nucleotide oxidases in human polymorphonuclear leukocytes. In the presence of strong base the oxidized forms of the pyridine nucleotides form a highly fluorescent product. The small amounts of NAD(P) formed by the oxidase reactions can be determined with great sensitivity. This method has been compared to the radioisotopic assay for NADPH oxidation. Both methods gave essentially the same results in terms of nanomoles NADP produced by control, resting, and phagocytizing samples.Both NADPH and NADH oxidase activities were insensitive to cyanide. NADPH oxidation had a pH optimum of 5.5, while that for NADH appeared to be 6.0. Granules isolated from phagocytizing cells routinely showed more activity toward both substrates (two to threefold) than granules from resting cells. Both activities were located primarily in a granule fraction prepared by differential centrifugation. Oxidation of NADPH was routinely four to five times that of NADH at all except very high substrate levels. Measurable NADH oxidation was rarely seen below 0.80 mM NADH, while NADPH oxidation was easily measurable at 0.20 mM. One patient with chronic granulomatous disease was studied. At low substrate levels, there was no activity toward either substrate in granules isolated from either resting or phagocytizing cells of this patient, while granules isolated from normal control cells showed substantial activity at these substrate levels.Purification of the activities has been initiated with linear sucrose gradients. Both activities co-sediment to

NAD(P)H oxidase activity in human neutrophils stimulated by phorbol myristate acetate.

“…The primary oxidase involved in this reaction has been reported to use NADH (19) or NADPH (12,20) preferentially. It has been assigned a pH optimum of 5.5-6.0 (7,(21)(22)(23) or near neutrality (20,24). It has been localized to the plasma membrane (25)(26)(27), or to other cellular organelles (7,24).…”

“…It has been assigned a pH optimum of 5.5-6.0 (7,(21)(22)(23) or near neutrality (20,24). It has been localized to the plasma membrane (25)(26)(27), or to other cellular organelles (7,24). Suggestions that its activation involves allosteric phenomena have been made (21), but not confirmed.…”

“…Both approaches were used in the present study. We began by applying a two-step procedure, devised by Auclair et al (28)(29)(30) and Iverson et al (7), that involves incubating GRF with NAD(P)H under specified conditions, followed by the measurement of NAD(P)+ fluorometrically (14). We found that two major and distinct processes caused NADPH to be oxidized to NAD(P)+ in step 1.…”

“…Under such circumstances, nonenzymatic secondary oxidation of NAD(P)H by enzymatically generated O°obscured the primary oxidase reaction. Unless otherwise noted, our subsequent experiments were performed at pH 7 and in the presence of SOD. Linearity of NAD(P)+ production with incubation time and granule concentration under these conditions are shown in Fig.…”

Subcellular localization of the superoxide-forming enzyme in human neutrophils.

“…Besides these more indirect approaches, attempts have been made by several investigators to localize the enzyme responsible for the respiratory burst in subcellular fractions of neutrophils (27)(28)(29). Variously, the activity has been attributed to the azurophil granule (27); to very dense particles distinct from azurophil granules which had hitherto not been described (28); and, depending upon the assay conditions, to the plasma membrane, the azurophil granule, or to several subcellular organelles (29).…”