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

Abstract

A fluormetric 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 had been initiated with linear sucrose gradients. Both activities co-sediment to a very dense region of the gradient, a region different from that in which membrane or azurophil granules usually equilibrate. The peak gradient fractions show a 10-30-fold increase in specific activity over comparable granule fractions. These data suggest that the oxidase activities are associated with one enzyme that has different affinities for the two substrates ans support the contention that the oxidation of NADPH is responsible for the metabolic burst accompanying phagocytosis in human PMNL.