Activation of a NADPH Oxidase From Horse Polymorphonuclear Leukocytes in a Cell- Free System

Abstract

A postnuclear cell fraction from resting horse polymorphonuclear (PMN) leukocytes incubated with fatty acid-salt ions such as oleate or linoleate generated a NADPH-dependent oxygen consumption and superoxide production. Oxidative activity was negligible or absent in the postnuclear fraction from mononuclear leukocytes, p-chloromercuribenzene sulfonic acid-treated granulocytes, and granulocytes from a patient with chronic granulomatous disease. Although consistently associated with the membrane fraction from resting PMN leukocytes, the superoxide-generating activity was shown to be dependent on a thus far unknown cytosolic constituent. The apparent K_m's for NADPH and NADH (66 and 1,600 µM, respectively), the pH optimum for the reaction (7.0), the cyanide insensitivity, and transient nature of the reaction together with the stoichiometric relationship between oxygen uptake and NADPH oxidation led to the conclusion that in the presence of cytosol a cell-free latent respiratory burst oxidase can be converted into an active enzyme by interaction with oleate micelles.